Human liver chimeric non-human animal with deficient p450 oxidoreductase and methods of using same

ABSTRACT

The present disclosure provides a chimeric non-human animal comprising human hepatocytes, methods for preparing the chimeric non-human animal comprising human hepatocytes and methods of utilizing the chimeric non-human animal comprising human hepatocytes to screening and identifying metabolites for any type of drugs, typically small molecule drugs, which might affect human liver functions and any other bodily function.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to, and the benefit of, U.S. Provisional Application No. 62/355,102, filed on Jun. 27, 2016 and U.S. Provisional Application No. 62/509,942, filed on May 23, 2017. The entire content of each of these applications is incorporated herein by reference in their entireties

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The contents of the text file name “KARL-001-WO_ST25,” which was created on Jun. 20, 2017 and is 67 KB in size are hereby incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

Only one out of ten drugs in development gets approved for clinical use. The majority fails during clinical trials due to inefficacy or toxicity in humans. The lack of experimental animal models to accurately predict human xenobiotic metabolism is a significant limitation, which jeopardizes human lives and drives drug development costs. Hence, there is a compelling need to develop better preclinical tools. The present disclosure solves these needs in the art by providing a human liver chimeric non-human animal model and methods of using the human liver chimeric non-human animal model to predict human specific drug metabolism.

SUMMARY OF THE INVENTION

The present disclosure provides a method for preparing a chimeric non-human animal comprising human hepatocytes, the method comprising: (a) providing a non-human animal comprising a reduction or deletion of NADPH-P450 oxidoreductase (Por) gene resulting in reduced or absent expression of Por protein; and (b) transplanting human hepatocytes into the non-human animal.

The non-human animal can comprise reducing or deleting the Por gene resulting in reduced or absent expression of Por protein. The reduced or deleted Por gene can be a conditional knockdown or knockout of the Por gene. The reduced or deleted Por gene can be the result of a mutation, a transgene, treatment with an exogenous substance or somatic genome engineering, including a CRISPR (Clustered regularly interspaced short palindromic repeats) system. The somatic genome engineering comprises Guide RNA (gRNA) and Caspase 9 (Cas9).

The non-human animal can comprise a floxed allele of the Por gene, and wherein the non-human animal is provided with a Cre recombinase sufficient to produce a conditional knockout of the Por gene. The non-human animal comprising the floxed allele of the Por gene can be provided with at least a first dose of a virus that encodes Cre recombinase. The non-human animal can be provided with at least a second dose of a virus that encodes Cre recombinase. The non-human animal can comprise the floxed allele of the Por gene is crossed with a transgenic non-human animal strain expressing Cre recombinase.

In a one aspect, the method of the present disclosure comprises (a) providing a non-human animal comprising a foxed allele of the Por gene with a first does of a virus that encodes Cre recombinase; (b) transplanting human hepatocytes into the non-human animal; and (c) providing the non-human animal with a second dose of a virus that encodes Cre recombinase. Steps (a) and (b) can occur sequentially or simultaneously.

The non-human animal can further comprise a reduction or deletion of at least one additional gene encoding an enzyme involved in drug metabolism. The at least one additional enzyme can be a phase II drug enzyme. In one aspect, the non-human animal can further comprise a reduction or deletion of UDP-glucose 6-dehydrogenase (UGDH) gene, a reduction or deletion of Glutathione synthetase (GSS) gene, or a combination thereof.

The reduction or deletion the UGDH gene can result in reduced or absent expression of UGDH protein. The reduction or deletion the GSS gene can result in reduced or absent expression of GSS protein. The reduced or deleted UGDH gene can be a conditional knockdown or knockout of the UGDH gene. The reduced or deleted GSS gene can be a conditional knockdown or knockout of the GSS gene. The reduced or deleted UGDH or GSS gene can be the result of a mutation, a transgene, treatment with an exogenous substance or somatic genome engineering, including a CRISPR (Clustered regularly interspaced short palindromic repeats) system. The somatic genome engineering comprises Guide RNA (gRNA) and Caspase 9 (Cas9).

The non-human animal can be selected from the group consisting of primate, bird, mouse, rat, fowl, dog, cat, cow, horse, goat, camel, sheep and pig. In a preferred aspect, the non-human animal is a mouse.

The non-human animal comprising a reducing or deleting the Por gene can be selected from the group consisting of (i) the FRG (Fah^(−/−)/Rag2^(−/−)/Il2rg^(−/−)) non-human animal, (ii) a transgenic urokinase type plasminogen activator (uPA) non-human animal, which overexpress uPA under an inducible promoter, preferably a liver-restricted albumin promoter, (iii) the thymidine kinase-NOD/Shi-scid/IL-2Rγ^(null) (TK-NOG) non-human animal, which is a immunodeficient NOG non-human animal with transgenic expression of thymidine kinase under control of liver-restricted promoter, (iv) a non-human animal expressing an inducible Caspase 8 in the liver, and (v) a non-human animal expressing an inducible Caspase 9 in the liver.

The present disclosure also provides a chimeric non-human animal, offspring thereof, or a portion thereof, which has a chimeric liver comprising human hepatocytes, prepared by any method disclosed herein.

The chimeric non-human animal can be immunodeficient. The chimeric non-human animal substantially lacks autogenous hepatocytes. Human hepatocytes can account for any percentage of human chimerism greater than about 1%, for example at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% of all hepatocytes in the chimeric liver of the chimeric non-human animal. A “non-human animal” can be amphibian, reptile, avian, or a non-human mammal. The non-human animal can be e.g., any non-human mammal, e.g., primate, bird, mouse, rat, fowl, dog, cat, cow, horse, goat, camel, sheep or pig. In a preferred aspect, the non-human animal is a mouse.

In one aspect, the present disclosure provides a method for preparing a chimeric non-human animal comprising human hepatocytes, the method comprising steps of: (a) providing a non-human animal that allows its liver to be repopulated with human hepatocytes and comprising a non-functional NADPH-P450 oxidoreductase generated either by genome engineering or knockdown with exogenous agents such as genome engineering tools like CRISPR/Cas9 or floxed allele of the NADPH-P450 oxidoreductase (Por) gene with a first dose of a virus that encodes Cre recombinase or a Cre transgenic animal, thereby producing a conditional knockout of the Por gene; (b) transplanting human hepatocytes into the non-human animal; and (c) providing the non-human animal with a second dose of the virus that encodes Cre recombinase. The chimeric non-human animal can substantially lack autogenous or endogenous hepatocytes and instead comprising human hepatocytes. Steps (a) and (b) can occur sequentially or simultaneously. Any non-human animal comprising mutations and/or transgenes that allow its liver to be repopulated with human hepatocytes may be used in combination with the floxed or deleted allele of the NADPH-P450 oxidoreductase (Por) gene or functional inactivation of the Por protein. In aspects, the non-human animal comprising mutations and/or transgenes that allow its liver to be repopulated with human hepatocytes is (i) the FRG (Fah^(−/−)/Rag2^(−/−)/Il2rg^(−/−)) non-human animal, (ii) a transgenic uPA non-human animal, which overexpress urokinase type plasminogen activator (uPA) in the liver under an inducible promoter and/or preferably a liver-restricted albumin promoter, (iii) the TK-NOG non-human animal, which is a immunodeficient NOG non-human animal with transgenic expression of thymidine kinase under control of liver-restricted albumin promoter, (iv) a non-human animal expressing an inducible Caspase 8 in the liver, or (v) a non-human animal expressing an inducible Caspase 9 in the liver (vi) a non-human animal expressing human heparin-binding epidermal growth factor-like receptor (HB-EGF)-like receptors under the control of a liver cell-specific albumin promoter (alb-TRECK). A “non-human animal” can be amphibian, reptile, avian, or a non-human mammal.

In one aspect, the present disclosure provides a method for preparing a chimeric mouse substantially lacking murine hepatocytes and instead comprising human hepatocytes, comprising steps of: (a) providing a mouse that allows its liver to be repopulated with human hepatocytes and comprising a non-functional NADPH-P450 oxidoreductase generated either by genome engineering by CRISPR/Cas9 mediated deletion or knockdown with exogenous agents or foxed allele of the NADPH-P450 oxidoreductase (Por) gene with a first dose of a virus that encodes Cre recombinase or a Cre transgenic mouse, thereby producing a conditional knockout of the Por gene; (b) transplanting human hepatocytes into the mouse; and (c) providing the mouse with a second dose of the virus that encodes Cre recombinase. Steps (a) and (b) can occur sequentially or simultaneously. Any mouse that allow its liver to be repopulated with human hepatocytes may be used in combination with the floxed allele of the NADPH-P450 oxidoreductase (Por) gene or somatic gene deletion or reduction or inactivation of the Por gene, respectively protein. In aspects, the mouse that allow its liver to be repopulated with human hepatocytes is (i) the FRG (Fah^(−/−)/Rag2^(−/−)/Il2rg^(−/−)) mouse, (ii) a transgenic uPA mouse, which overexpress urokinase type plasminogen activator (uPA) under an inducible promoter, preferably a liver-restricted albumin promoter, (iii) the TK-NOG mouse, which is a super immunodeficient NOG mouse with transgenic expression of thymidine kinase under control of liver-restricted albumin promoter, (iv) a mouse expressing an inducible Caspase 8 in the liver, (v) a mouse expressing an inducible Caspase 9 in the liver or (vi) a mouse expressing human heparin-binding epidermal growth factor-like receptor (HB-EGF)-like receptors under the control of a liver cell-specific albumin promoter (alb-TRECK).

The present disclosure also provides a method for screening and identifying metabolites for any type of drugs, typically small molecule drugs, that might affect human liver functions but also any other function of the body, comprising: (a) administering a test substance to the chimeric non-human animal of the present disclosure; (b) measuring one or more values in the chimeric non-human animal to which the test substance is administered in (a); and (c) selecting a test substance that causes an increase or an decrease in one or more values measured in (b), compared with the one or more values measured in a chimeric non-human animal to which no test substance is administered or a chimeric non-human animal without deletion of the Por gene or a non-human animals without human chimerism. Preferably, the one or more values are selected from but not limited to the group consisting of a metabolite of the test substance, human albumin concentration, body weight curve, liver-weight-to-body-weight ratio, total albumin level, total protein level, Alanine Aminotransferase (ALT) level, Aspartate Aminotransferase (AST) level, and total bilirubin level, creatinine, Blood Urea Nitrogen (BUN), troponine, blood count, TSH and histological assessment for pathologies in the human and non-human organs. A “non-human animal” can be amphibian, reptile, avian, or a non-human mammal. The non-human animal can be e.g., any non-human mammal, e.g., primate, bird, mouse, rat, fowl, dog, cat, cow, horse, goat, camel, sheep or pig. Preferably, the non-human animal is a mouse.

The present disclosure further provides a method for screening for a substance that affects human liver functions, comprising: (a) administering a test substance to the chimeric mouse of the present disclosure; (b) measuring one or more values in the chimeric mouse to which the test substance is administered in (a); and (c) selecting a test substance that causes an increase or an decrease in one or more values measured in (b), compared with the one or more values measured in a chimeric mouse to which no test substance is administered. Preferably, the one or more values is selected from the group consisting of a metabolite of the test substance, human albumin concentration, body weight curve, liver-weight-to-body-weight ratio, total albumin level, total protein level, ALT level, AST level, and total bilirubin level, histological assessment for pathologies in the human and non-human organs.

The present disclosure also provides a method for evaluating the toxicity of a test substance against human hepatocytes, comprising: (a) administering a test substance to the chimeric non-human animal of the present disclosure; (b) measuring one or more indicators in the chimeric non-human animal to which the test substance is administered in (a); and (c) evaluating the effect of the test substance on human hepatocytes using, one or more indicators measured in (b), compared with the one or more indicators measured in a chimeric non-human animal to which no test substance is administered. Preferably, the one or more indicators is selected from the group consisting of an increase or a decrease in any one or more of a metabolite of the test substance, human albumin concentration, body weight curve, liver-weight-to-body-weight ratio, total albumin level, total protein level, ALT level, AST level, and total bilirubin level, histological assessment for toxicity in the human and non-human organs. A “non-human animal” can be amphibian, reptile, avian, or a non-human mammal. The non-human animal can be e.g., any non-human mammal, e.g., primate, bird, mouse, rat, fowl, dog, cat, cow, horse, goat, camel, sheep or pig. Preferably, the non-human animal is a mouse.

Throughout the specification the word “comprising,” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values provided herein are modified by the term “about.”

While the disclosure has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the disclosure, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. Genbank and NCBI submissions indicated by accession number cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

The above and further features will be more clearly appreciated from the following detailed description when taken in conjunction with the accompanying drawings.

FIG. 1A-D shows the generation of the PIRF strain and deletion of the murine P450 (Por) oxidoreductase. FIG. 1A is a schematic representation of deleted and transgenic loci in the PIRF strain. FIG. 1B is a graph showing qPCR of Por mRNA upon intravenous injection of adenovirus expressing the CRE recombinase (Adeno-Cre). FIG. 1C is a series of immunostaining photographs for Por demonstrating a gradient across the hepatic acinus with higher pericentral (cv) and lower periportal (pv) expression. Upon injection with Adeno-CrePor is barely detectable. FIG. 1D is a photograph of a Western blot showing the almost complete disappearance of Por protein.

FIG. 2A-E shows the humanization of the PIRF strain and gene expression profiling upon deletion of murine P450 oxidoreductase (Por). FIG. 2A is a series of immunostaining photographs showing humanized PIRF and FRG mice for murine Por (mPor) and human nuclei (hNuc) after injection of Adeno-Cre (2.2×10¹⁰ pfu/mouse) once (1×) or twice (2×). Counterstaining in merged picture using DAPI. FIG. 2B is a schematic showing the experimental outline for murine and human transcriptomics from chimeric livers with or without Por deletion. FIG. 2C is a graph showing the murine cytochrome mRNA originating from livers of the PIRF model. FIG. 2D is a graph showing the human cytochrome mRNA originating from livers of the PIRF model. FIG. 2E is a graph showing the comparison gene expression of the main drug metabolizing human cytochromes in humanized, Por-deleted PIRF (Hu-PIRF 2×) mice with the original, isogenic human hepatocytes. Gene expression has been normalized to three murine respectively human housekeeping genes (PSMB2, PSMB4 and RAB7A resp. Rab7²⁵). PIRF; Por^(c/c)/Il2rg^(−/−)/Rag2^(−/−), Fah^(−/−)FRG; Fah^(−/−)/Rag2^(−/−)/Il2rg^(−/−).

FIG. 3A-E shows xenobiotic metabolism in humanized PIRF mice. FIG. 3A is a graph showing the selection of abundant and decreased gefitinib metabolites upon murine P450 oxidoreductase (Por) deletion in non-humanized PIRF mice using mass spectrometry in the murine feces within 24 hours after intravenous injection of gefitinib. FIG. 3B is a schematic showing gefitinib metabolites and know modifications. FIG. 3C is a graph showing that murine Por-deleted, human liver chimeric PIRF (Hu-PIRF 2×) mice and control groups show the most abundant human metabolite, M4. FIG. 3D is a graph showing that murine Por-deleted, human liver chimeric PIRF (Hu-PIRF 2×) mice and control groups show the human specific metabolite M28. PIRF; Por^(c/c)/Il2rg^(−/−)/Rag2^(−/−), Fah^(−/−)FRG; Fah^(−/−)/Rag2^(−/−)/Il2rg^(−/−). * p<0.05 using non-parametric Mann-Whitney test. FIG. 3E is a graph is a graph showing a mass spectrometry analysis of PIRF liver homogenates 30 min after injection with atazanavir, a retroviral therapeutic. A major human metabolite (M15) is shown. The overall abundance of metabolites from atazanavir or gefitinib was set as 100% in each sample. The data are expressed as mean. PIRF; Por^(c/c)/Il2rg^(−/−)/Rag2^(−/−)/Fah^(−/−), FRG; Fah^(−/−)/Rag2^(−/−)/Il2rg^(−/−) * p<0.05 using non-parametric Mann-Whitney test.

FIG. 4A-B shows a schematic of an exemplary probe of the present disclosure. FIG. 4A is a schematic showing the design of the targeting vector and modified Por locus. FIG. 4B is a photograph of Southern blotting with three probes demonstrating proper targeting of ESC.

FIG. 5 shows beta galactosidase (lacZ) expression from the POR-lacZ allele. X-gal staining of heterozygous mouse embryo and liver demonstrate expression of the galactosidase from the Por-lacZ allele.

FIG. 6A shows a scheme of Il2 rg, Rag2 and Fah genes showing the gRNA location (in color) and the primers used to genotype the mice. FIG. 6B shows an electrophoresis gel of the PCR products obtained from genotyping. Lane 1: PCR bands using external (Fw and Rv) primers. For Rag2 and Fah heterozygotes, a wild type (arrow) and deleted (arrowhead) allele can be detected. Il2 rg is an X-linked gene and no founder heterozygote females were generated. Homozygotes mice for Il2 rg, Rag2 and Fah show a single deleted allele's band of 460 bp, 530 bp and 200 bp, respectively. Lane 2: PCR bands using one of the external (Fw or Rev) and the internal (Int) primer located between the two gRNA sites. Only heterozygotes have a clear PCR band formed from the wild type allele. Fah and Rag2 homozygotes show multiple unspecific bands while Il2 rg homozygotes do not produce any band.

FIGS. 7A and 7B show the spectrum of genomic deletions in the Il2-rg, Rag2 and Fah gene. FIG. 7A is a schematic showing CRISPR/Cas9 injected zygotes of conditional Por−/− mice sequenced to determine of deletion of DNA. FIG. 7B is a schematic showing CRISPR/Cas9 injected zygotes of conditional Por−/− mice sequenced to determine of deletion of amino acids.

FIG. 8 shows lipid phenotype in the liver of PIRF mice upon deletion of the Por gene using an adenovirus expressing the CRE recombinase. Two weeks after injection of the adenovirus hepatocytes start to accumulated lipids. Oil-red-O stained livers have previously been validated for efficient deletion (upper panel) respectively expression (lower panel) of the Por gene (not shown).

FIG. 9 shows clonal expansion of Por expressing hepatocytes in non-humanized PIRF mice. Mice were injected intravenously with adenovirus expressing CRE recombinase deleting the Por gene (POR^(fl/fl)).

FIG. 10 shows the experimental setup for drug studies in humanized and non-humanized control mice. Humanized (Hu) and not-humanized PIRF and FRG ice were injected once (1×, before transplantation of human hepatocytes) or twice (2×, before and after reaching high human chimerism) before doing drug studies. Injected adenovirus expresses the CRE-recombinase, which leads to deletion of the Por gene in the PIRF, but not in the FRG strain (control). Orange; murine drug metabolism, white; inhibited murine drug metabolism, blue; human drug metabolism.

FIGS. 11A and 11B show that the conditional KO of POR can also be generated using a transgenic animal, which carries an expression cassette (albumin promoter) of the CRE recombinase. FIG. 11A shows that POR can be detected in immunofluorescence with the control group where only one allele carries a foxed POR sequence the other one the wild-type POR (POR^(c/+)). In FIG. 11B, both POR alleles are floxed (POR^(c/c)), leading to an almost complete deletion of the POR gene and a barely detectable POR protein. POR (green) and nuclei (blue, DAPI).

FIG. 12 depicts the expression of P450 oxidoreductase in humanized PIRF mice. FIG. 12A is a bar graph showing qPCR of human and murine specific Por normalized to human and murine Gapdh, respectively. FIG. 12B is a western blot image showing murine Por and β-actin of liver samples from the same humanized mice.

FIG. 13 depicts Gefitinib metabolites upon murine P450 oxidoreductase (Por) deletion. FIG. 13A is a bar graph showing deletion by adenoviral delivery of CRE. FIG. 13B is a bar graph showing by crossing of Por^(c/c) with Alb-Cre mice, generating an Alb-Cre/Por^(c/c) strain.

FIG. 14 depicts the deletion of murine P450 oxidoreductase by crossing Por^(c/c) with an Alb-Cre transgenic mouse. FIG. 14A is a confocal immunostaining image showing complete Por deletion. FIG. 14 b is a western blot image showing Por protein liver samples from a control Por^(c/c) mouse and three different Alb-Cre/Por^(c/c) mice. FIG. 14C is a bar graph showing qPCR of murine Por mRNA levels.

FIG. 15 is a bar graph showing Gefitinib metabolite M28 in feces.

FIG. 16 is a pair of bars graphs showing Gefitinib metabolite M28 in the (A) serum and (B) urine of hu-PIRF-2× mice and control groups.

FIG. 17 is a Southern blot image of targeted ESCs. FIG. 17A shows the use of a 5′probe, FIG. 17B shows the use of a 3′ probe and FIG. 17C shows the use of a neomycin probe.

FIG. 18 is a Western blot image showing murine Por and Gadph from PIRF mice injected with Adeno-CRE.

FIG. 19A-B is a Western blot image showing murine Por and Gadph from Humanized PIRF mice (a) and Por^(c/c) and (b) Por^(c/c)/Alb-CRE mice.

FIG. 20 is an H&E stain of liver lobe four weeks after transduction with adenovirus showing macro- and microvesicular steatosis. Left picture is a higher magnification of boxed area on right FIG. 21A-B shows gene therapy vector design for liver-specific deletion by genome engineering of drug metabolizing enzymes in humanized mice. A. Two vector design: S. pyogenes Cas9 under the control of CMV promoter in an adenoviral vector (Ad), and Adeno-Associated Virus (AAV) expressing a sgRNA targeting a drug metabolizing enzyme and co-expression of GFP on the same construct. FIG. 21B. Single vector design: S. aureus Cas9 and sgRNA can be delivered on the same AAV vector (4.85 kb). HA, HA-epitope.

FIG. 22 shows simultaneous deletion of murine P450 oxidoreductase (Por) and other murine enzymes involved in drug metabolism in humanized mice by somatic genome engineering. Humanized FRG mice (human albumin in murine serum >2 mg/ml) have been injected with Adeno-Associated Virus (AAV, serotype 8) expressing sgRNA targeting an early exon of murine Por, UDP-glucose 6-dehydrogenase (Ugdh) or the glutathione synthetase (Gss) gene (see gene therapy vector design, FIG. 21 ). AAVs have been injected (2×10¹¹ GC/AAV/mouse) 1 week before injection of Adenovirus expressing Cas9 (7×10⁹ pfu/Ad/mouse). Control mice have been injected with adenoviral vector only (lower row). Shown are serial sections of a representative humanized area with immunostaining for human specific Pre-ALB (transthyretin), Por, Ugdh, Gss, hALB and GFP, the latter being expressed from the AAV gene therapy vector (see gene therapy vector design, FIG. 21 ). Bar represents 50 μm. ALB, Albumin.

FIG. 23 shows genomic deletion of por by CRISPR/Cas9: Wild type and humanized FRG mice (human albumin in murine serum >2 mg/ml) are injected with two Adeno-Associated Virus (AAV, serotype 8) expressing sgRNA targeting consecutive early exons of murine Por as well as S. aureus Cas9 (see gene therapy vector design, FIG. 21 ). AAVs are injected (2×10¹¹ GC/AAV/mouse). Control mice are injected with adenoviral vector expressing Cas9 only (7×10⁹ pfu/Ad/mouse). Shown are PCR amplifications of the region spanning the two nearby targeting sites. Upon CRISPR/Cas9 mediated cutting of DNA on both target sites results in a deletion of the por gene leading to a smaller PCR amplicon.

FIG. 24 shows humanized PIRF mouse with transgenic Alb-CRE and deletion of other murine enzymes involved in drug metabolism. Por was deleted by expression of CRE, but instead of adenoviral CRE, this PIRF mouse carries an Alb-CRE sequence within the murine genome. After humanization, mice were injected with Adeno-Associated Virus (AAV, serotype 8) expressing sgRNA targeting an early exon UDP-glucose 6-dehydrogenase (Ugdh) or the glutathione synthetase (G s s) gene (see gene therapy vector design, FIG. 21 ). AAVs are injected (2×10¹¹ GE/AAV/mouse) 1 week before injection of Adenovirus expressing Cas9 (7×10⁹ GE/Ad/mouse). Shown are serial sections of a representative humanized area with immunostaining for human specific Pre-ALB (transthyretin), Por, Ugdh or Gss. Bar represents 50 μm. ALB, Albumin.

FIG. 25A-D shows troglitazone Phase II metabolites detected in liver of humanized and non-humanized FRG mice with and without Por and Ugdh deletion. Percentage of liver glucuronidated and sulfated metabolites detected in humanized livers 2 hours after i.p injection of troglitazone (600 mg/kg). FIG. 25A. Humanized FRG mice. FIG. 25B. Humanized FRG mice after murine Por and Ugdh deletion. FIG. 25C. Non-humanized FRG mice. FIG. 25D. Non-humanized FRG mice with Por and Ugdh genes deleted. Sulfate metabolites of troglitazone in red and glucuronide conjugates in blue.

DETAILED DESCRIPTION OF THE INVENTION

Human liver chimeric mice have been recently introduced to predict human xenobiotic metabolism and toxicity. Despite their potential, the remaining murine liver, containing an expanded set of P450 cytochromes, makes it difficult to accurately predict human drug metabolism. Therefore, the present disclosure provides a conditional knock-out mouse of the NADPH-P450 oxidoreductase (Por) gene, which is the only electron donor for all murine cytochromes and if deleted, embryonically lethal¹, thereby allowing a functional inactivation of all murine cytochromes.

Any mouse comprising mutations and/or transgenes that allow its liver to be repopulated with human hepatocytes may be used in combination with the conditional knock-out allele or other genomic deletion of the NADPH-P450 oxidoreductase (Por) gene. In embodiments, the mouse comprising mutations and/or transgenes that allow its liver to be repopulated with human hepatocytes is (i) the FRG (Fah^(−/−)/Rag2^(−/−)/Il2rg^(−/−)) mouse, (ii) a transgenic uPA mouse, which overexpress urokinase type plasminogen activator (uPA) under an inducible promoter, preferably a liver-restricted albumin promoter, (iii) the TK-NOG mouse, which is a immunodeficient NOG mouse with transgenic expression of thymidine kinase under control of liver-restricted albumin promoter, (iv) a mouse expressing an inducible Caspase 8 in the liver, (v) a mouse expressing an inducible Caspase 9 in the liver or (vi) a mouse expressing human heparin-binding epidermal growth factor-like receptor (HB-EGF)-like receptors under the control of a liver cell-specific albumin promoter (alb-TRECK). Using such mice and an adenoviral or transgenic strategy expressing CRE, an almost complete deletion of the murine Por gene can be generated leading to an exclusive human cytochrome metabolism.

In the uPA-SCID mouse (Rhim et al 1994; Tateno et al. 2004), the genetic cause of mouse hepatocyte ablation is uroplasminogen activator (uPA); the mouse is in the SCID immune deficient background or Rag2 (or Rag1)−/− and/or Il2rg−/− all leading to the ability to transplant and engraft human hepatocytes.

In the FRG mouse (Azuma et al 2007⁷, Bissig et al 2007⁵), the genetic cause of mouse hepatocyte ablation is fumarylacetoacetate hydrolase deficiency and mouse hepatocyte ablation is controlled by ±NTBC and/or ±low tyrosine diet; the mouse is in the Il2rg−/− and Rag2−/− background. The FRG mouse combines immune-deficiency-mediating mutations, in the recombination activating gene 2 (Rag2) and the gamma chain of the interleukin 2 receptor (Il2rg), with a functional knockout of the fumarylacetoacetate hydrolase (Fah) gene (Azuma et al 2007⁷, Bissig et al 2007⁵), The latter gene codes for an enzyme in the tyrosine catabolic pathway and its mutation leads to an intracellular accumulation of a toxic inter-mediate in hepatocytes. Unlike the uPA/SCID model, the onset and severity of hepatocellular injury in FRG mice is controllable through the administration and withdrawal of the protective drug 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC), which blocks an upstream enzyme in the tyrosine path-way and thereby prevents accumulation of the toxic intermediate.

In the TK-NOG mouse (Hasegawa et al 2011), the genetic cause of mouse hepatocyte ablation is the herpes simplex virus thymidine kinase and mouse hepatocyte ablation is controlled by ±ganciclovir; the mouse is in the Il2rg−/− and SCID background. Mouse hepatocyte ablation in this TK-NOG model was achieved through the liver-specific expression of the herpes simplex virus 1 thymidine kinase (HSVtk) in severely immunodeficient NOG mice and administration of ganciclovir (GCV), utilizing the fact that HSVtk converts the otherwise nontoxic GCV into a toxic intermediate.

In the AFC8 mouse (Washburn et al 2011), the genetic cause of mouse hepatocyte ablation is a FK508-capsae 8 fusion and mouse hepatocyte ablation is controlled by ±AP20187; the mouse is in the Il2rg−/− and Rag2−/− background.

In the Alb-TRECK/SCID mouse (Zhang et al 2015), the genetic cause of mouse hepatocyte ablation is the human heparin-binding EGF-like receptor and mouse hepatocyte ablation is controlled by ±Diphtheria toxin; the mouse is in the SCID immune deficient background.

Sheer and Wilson, 2015 compares major features of various different liver humanized models and process of liver reconstitution in the most frequently used models to date. This reference is incorporated by reference in its entirety.

The present disclosure also provides methods of utilizing the humanized, murine Por deficient mice to predict human drug metabolism. In an embodiment, the FRG mouse and the conditional Por−/− mouse was combined to generate the PIRF (Por−/−Il2rg−/−/Rag2−/−/Fah−/−) strain, which allows repopulation with human hepatocytes. Homozygous PIRF mice are fertile and can be repopulated with human hepatocytes generating high human chimerism (>80% human).

Human p450 cytochrome clusters contain 57 putatively functional genes and 58 pseudogenes, while the mouse cytochrome clusters are greatly expanded accounting for 102 putatively functional genes and 88 pseudogenes². This makes accurate prediction of human drug metabolism in the mouse challenging. In addition hepatotoxicity together with hypersensitivity/cutaneous reactions have the poorest correlation with animal studies yet are the most common reasons for toxicity related termination of drugs in clinical development³.

Since the liver is the main organ for drug metabolism, human liver chimeric mice are increasingly used for xenobiotic studies⁴⁻⁶. The shortcoming of humanized mice is the remaining murine liver tissue. It has been previously shown that even in mice that can achieve high human chimerism, the average humanization rate is 42%⁷. In order to functionally block the murine cytochrome metabolism, a conditional (floxed exon 3 and 4) knock-out of the NADPH-P450 oxidoreductase (Por) gene was generated by targeting mouse embryonic stem cells⁸ (FIG. 4 ). Injected blastocysts with properly targeted embryonic stem cells generated mice with germline transmission of the Por “knock-out first” allele⁹. Expression from the targeted Por locus using the lacZ expression cassette was confirmed in the embryo and adult liver (FIG. 5 ). The mice were then bred with a flippase expressing strain¹⁰ to generate a CRE recombinase conditional Por knock-out strain. Homozygous zygotes from this strain were injected with the bacterial type II Clustered Regularly-Interspaced Short Palindromic Repeats/Cas9 (CRISPR-Cas9) system¹¹⁻¹³ targeting simultaneous deletion of critical exons of the Il2-rg and Rag2 and Fah gene (FIG. 6 ) to generate the PIRF strain (FIG. 1A). Homozygous PIRF mice are immune deficient (T-, B- and NK-cell deficient), but healthy and fertile. Since adenoviral gene therapy vectors efficiently transduce hepatocytes in vivo, the Por gene was deleted using an adenovirus coding the CRE recombinase (Adeno-CRE). Increasing doses (2.2×10⁸⁻¹⁰ per mouse) of the virus were injected intravenously into PIRF mice. Quantitative RT-PCR of the FOR mRNA in liver revealed efficient deletion only at high doses (FIG. 1B). Immunostaining for POR (FIG. 1C) confirmed these findings, while a minimal residual signal could be detected by Western blotting even at the highest dose used (FIG. 1D). POR-deleted PIRF mouse livers accumulated lipids starting two weeks after adenoviral transduction (FIG. 7 ) similar to a previously reported liver specific Por deletion¹⁴.

To generate human specific P450 cytochrome metabolism, human liver chimeric mice were generated by transplanting human hepatocytes^(7, 15, 16) into Por deleted PIRF mice. However, since a clonal expansion of residual Por expressing murine hepatocytes was observed in Adeno-Cre treated PIRF mice (FIG. 8 ), some humanized PIRF (Hu-PIRF) mice were injected with an additional dose of Adeno-Cre. Immunostaining revealed that only in double injected humanized PIRF (Hu-PIRF 2×) mice an almost complete deletion of the Por gene could be achieved (FIG. 2A).

Gene expression profiling was then performed comparing Hu-PIRF mice repopulated with the identical human hepatocytes with or without deletion of the Por (FIG. 2B). Expression of the murine P450 cytochromes was clearly altered for half of the genes: 14 cytochromes were upregulated >1.5-fold and 18 cytochromes downregulated <0.5-fold (FIG. 2 c ). The expression profiles of these murine cytochromes were comparable to previous work in non-humanized mice (Table 1). Table 1 shows the comparison of murine gene expression profiles of chimeric livers to previously published non-humanized mice. Gene expression of conditional (Alb-Cre) Por KO mice have been quantified by microarray analysis (Weng et al. 2005 J Biol Chem 280, 31686-31698 (2005)). Here, RNA-Seq was used to compare the gene expression (FIG. 2B) in humanized livers transduced with Adeno-Cre and Adeno-GFP. Table 1 lists all previously published cytochromes with values (fold changes) compared to the herein-described data set. Multiple numbers represent multiple sets of microarray probes.

TABLE 1 Comparison of murine gene expression profiles of chimeric livers to previously published non-humanized mice. Change Fold (Por-deleted/ Por non-deleted mice) Murine P450 Present Weng Cytochromes Disclosure et al. 2005 Cyp2a4 1.4  4.5 Cyp2a5 1.3  4.5 Cyp2b10 12.4 15.8/16.3/9.1 Cyp2c39 1.8  1.4 Cyp2c55 14.6 17.2 Cyp4a10 3.7 0.3/0.7 Cyp7a1 4.6 3.1/4.9 Cyp7b1 1.6 0.2/0.3 Cyp26a1 6.7  3.5 Cyp51 0.6  2.2

In the same chimeric liver, all human P450 cytochromes were down regulated upon deletion of murine Por with the exception of CYP2C18 (FIG. 2D). Half of the human cytochromes were only slightly (<50%) reduced, and the other half including CYP3A4 and CYP2C19, were more significantly downregulated (>1.5-fold).

Not all human cytochromes take an important role in xenobiotic metabolism. From the 200 most transcribed drugs in the United States about three quarter are metabolized through P450 cytochromes, of which CYP3A4/5, 2C9, 2C19, 2D6 and 1A2 contribute to −95% of 17. These human cytochrome clusters were compared from chimeric livers (Hu-PIRF 2×) with the originating, isogenic primary hepatocytes after isolation from the donor liver. Expression levels were similar for most clusters and these important cytochromes robustly expressed in chimeric livers (FIG. 3D).

To validate utility of Hu-PIRF mice for human drug metabolism, the xenobiotic metabolism of gefetinib¹⁸, an inhibitor of epidermal growth factor receptor used against lung cancer and a variety of other cancers¹⁹, was studied. Gefetinib is primarily metabolized by the P450 cytochrome system including CYP3A4 and 2D6. New gefetinib metabolites were recently identified and demonstrated considerable differences between human and mouse liver microsomes²⁰. Gefetinib is excreted in the feces and less than 7% in the urine, irrespectively of dose, route or species^(21, 22). Therefore, the feces of non-humanized PIRF mice was analyzed for gefetinib metabolites during the first 24-hours after intravenous injection of gefetinib. Mass spectrometry revealed a reduction of several gefetinib metabolites upon deletion of the Por gene, implying a Por-dependent P450 cytochrome deficiency for these metabolites (FIG. 3A). The biggest and most relevant reduction was observed for 0-desmethyl gefitinib (M4, M523595), which is by far the most abundant metabolite in human feces while rodents produce many different metabolites including M4^(21, 22) (FIG. 3B). Therefore, the M4 metabolite was analyzed in murine Por-deleted and Por-expressing humanized and non-humanized control mice (FIG. 9 ). The highest level of M4 could be detected in murine Por-deficient Hu-PIRF mice, where human hepatocytes preferentially metabolize gefitinib to M4 and remaining murine hepatocytes are inhibited in their drug metabolism (FIG. 3C). Although murine hepatocytes preferentially produce other metabolites than M4, human specific metabolites were measured. M28 was the most abundant human metabolite, which could not be detected in the non-humanized control mice. Mass spectrometry again showed the highest level of this human specific metabolite in murine Por-deficient Hu-PIRF mice confirming a more human like metabolism in these mice (FIG. 3D). Human xenobiotic metabolism was also determined with another drug, however, this time using liver homogenates of PIRF mice. Using human and mouse microsomes, it was previously demonstrated that atazanavir metabolite M15 is a predominately human metabolite (See, Li, F et al., “CYP3A-mediated generation of aldehyde and hydrazine in atazanavir metabolism.” Drug Metab Dispos 39, 394-401. Mice were intravenously injected with the retroviral therapeutic and livers were harvested 30 min after injection. Results showed that M15 was 5.4-times elevated in POR-deleted humanized PIRF mice compared to non-deleted mice (FIG. 3E) again confirming optimized human drug metabolism in this novel mouse model.

Identification of human metabolites using current experimental animal models is a major challenge. Nevertheless, identification of reactive metabolites is crucial since they drive human drug toxicity^(23, 24). The novel humanized mouse model of the instant disclosure inhibits murine drug metabolism without impeding on the human metabolism. Murine Por-deficient humanization can be used in combination with other repopulation models like the transgenic uPA mouse and can identify more readily human specific metabolites for a greater benefit of drug safety.

Identification of mostly human or human specific metabolites is possible with the present disclosure irrespectively of toxicity. Toxicity may be present; however this is not always the case. For instance, as shown here, gefitinib did not cause any elevation of liver enzymes, yet mainly human metabolites were identified.

The present disclosure provides a method for preparing a chimeric mouse substantially lacking murine hepatocytes and instead comprising human hepatocytes, comprising steps of: (a) providing a mouse comprising a knockout mutation in each of the Il2-rg, Rag2, and Fah genes and a foxed allele of the NADPH-P450 oxidoreductase (Por) gene with a first dose of a virus that encodes Cre recombinase, thereby producing a conditional knockout of the Por gene or the knockout of the por gene using somatic genome engineering (CRIPSR/Cas9) and gene therapy vectors in Il2-rg, Rag2, and Fah deficient mice; (b) transplanting human hepatocytes into the mouse; and (c) providing the mouse with a second dose of the virus that encodes Cre recombinase. Steps (a) and (b) can occur sequentially or simultaneously.

The conditional knock-out POR alleles can also be generated by delivering CRE recombinase in any way known in the art. Non-limiting examples of Cre recombinase delivery include viral or non-viral gene therapy vectors. In one embodiment, the gene therapy vector is an adenovirus. Also considered are genetic delivery of Cre recombination, e.g., under a cell-, tissue-, or developmental-specific promoter or under an inducible promoter. Indeed, Cre recombinase can be activated in the murine liver in a transgenic animal with Cre expressed under the albumin or other liver specific promoter (FIG. 11 ).

The present disclosure also provides a chimeric mouse, offspring thereof, or a portion thereof, which has a chimeric liver comprising human hepatocytes. Preferably, the chimeric mouse, offspring thereof or a portion thereof is prepared by the methods of the present disclosure. The chimeric mouse can be immunodeficient.

In the present disclosure, examples of the chimeric mouse include portions of the mouse. The term “a portion(s) of the mouse” refers to, mouse-derived tissues, body fluids, cells, and disrupted products thereof or extracts therefrom, for example (the examples thereof are not particularly limited to them). Examples of such tissues include, but are not particularly limited to, heart, lungs, kidney, liver, gallbladder, pancreas, spleen, intestine, muscle, blood vessel, brain, testis, ovary, uterus, placenta, marrow, thyroid gland, thymus gland, and mammary gland. Examples of body fluids include, but are not particularly limited to, blood, lymph fluids, and urine. The term “cells” refers to cells contained in the above tissues or body fluids, and examples thereof include cultured cells, sperm cells, ova, and fertilized eggs obtained by isolation or culture thereof. Examples of cultured cells include both primary cultured cells and cells of an established cell line. Examples of the portions of the mouse also include tissues, body fluids, and cells at the developmental stage (embryonic stage), as well as the disrupted products or extracts thereof. In addition, an established cell line from the mouse of the present disclosure can be established using a known method (Primary Culture Methods for Embryonic Cells (Shin Seikagaku Jikken Koza (New Biochemical Experimental Lecture Series), Vol. 18, pages 125-129, TOKYO KAGAKU DOZIN CO., LTD., and Manuals for. Mouse Embryo Manipulation, pages 262-264, Kindai Shuppan)).

The mouse of the present disclosure can be an immunodeficient mouse. The immunodeficient mouse of the present disclosure can be used as a host mouse for transplantation of human hepatocytes. Examples of the “immunodeficient mouse” may be any mouse that does not exhibit rejection against hepatocytes (in particular, human hepatocytes) from a different animal origin, and include, but are not limited to, SCID (severe combined immunodeficiency) mice exhibiting deficiency in T- and B-cell lines, mice (NUDE mice) that have lost T cell functions because of genetic deletion of the thymus gland, and mice (RAG2 knockout mice) produced by knocking out the RAG2 gene by a known gene targeting method (Science, 244: 1288-1292, 1989).

Moreover, the present disclosure provides a chimeric mouse having human hepatocytes. The chimeric mouse of the present disclosure can be immunologically deficient. The chimeric mouse of the present disclosure can be prepared by transplanting human hepatocytes into an immunodeficient mouse of the present disclosure.

As human hepatocytes to be used for transplantation, human hepatocytes isolated from normal human liver tissue by a conventional method such as a collagenase perfusion method can be used. The thus separated hepatocytes can also be used by thawing after cryopreservation. Alternatively, the chimeric mouse hepatocytes, which are defined as the human hepatocytes separated by a technique such as a collagenase perfusion method from a chimeric mouse liver, in which mouse hepatocytes have been replaced by human hepatocytes, can be used in a fresh state, and the cryopreserved chimeric mouse hepatocytes are also available after thawing.

Such human hepatocytes can be transplanted into the liver via the spleen of a mouse of the present disclosure. Such human hepatocytes can also be directly transplanted via the portal vein. The number of human hepatocytes to be transplanted may range from about 1 to 2,000,000 cells and preferably range from about 200,000 to 1,000,000 cells. The gender of the mouse of the present disclosure is not particularly limited. Also, the age on days of the mouse of the present disclosure upon transplantation is not particularly limited. When human hepatocytes are transplanted into a young mouse (early weeks of age), human hepatocytes can more actively proliferate as the mouse grows. Hence, about 0- to 40-day-old mice after birth, and particularly about 8- to 40-day-old mice after birth are preferably used.

The transplanted human hepatocytes account for any percentage of human chimerism greater than about 1%, for example at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% of all hepatocytes in the chimeric liver of the chimeric non-human animal.

The present disclosure further provides a method for screening for a substance that affects human liver functions, with the use of the chimeric mouse of the present disclosure. An example of the method is an evaluation method comprising the following steps of: (a) administering a test substance to the chimeric mouse of the present disclosure; (b) measuring one or more values in the chimeric mouse to which the test substance is administered in (a); and (c) selecting a test substance that causes an increase or an decrease in one or more values measured in (b), compared with the one or more values of the chimeric mouse to which no test substance is administered.

Preferably, the one or more values are selected from the group consisting of the human albumin concentration, the body weight curve, the liver-weight-to-body-weight ratio, the total albumin level, the total protein level, the ALT level, the AST level, and the total bilirubin level, histological assessment for toxicity in the human and non-human organs.

Examples of the “test substance” in the method of the present disclosure are not particularly limited and include natural compounds, organic compounds, inorganic compounds, proteins, antibodies, peptides, and single compounds such as an amino acid, and nucleic acids, as well as compound libraries, expression products from gene libraries, cell extracts, cell culture supernatants, products of fermenting microorganisms, extracts from marine creatures, plant extracts, extracts from prokaryotic cells, extracts from eukaryotic single cells, and extracts from animal cells. These products may be purified products or crude products such as plant, animal, or microbial extracts. Also, a method for producing a test substance is not particularly limited. A test substance to be used herein may be a substance isolated from a natural product, synthesized chemically or biochemically, or prepared by genetic engineering techniques.

The above test substance can be adequately labeled and then used as necessary. Examples of labels include radiolabels and fluorescent labels. Examples of the test substance include, in addition to the above test samples, mixtures of a plurality of types of these test samples.

Examples of test samples include and are not limited to feces, urine, blood (and any blood product, e.g., whole blood, serum, and plasma), and tissue, e.g., liver tissue. Liver tissue may be derived from a sample of a liver (e.g., a biopsy or explant) or may be derived from a whole, intact liver, e.g., that has been harvested after a mouse has been sacrificed.

Examples of a method for administering a test substance to mice are not particularly limited. Such an administration method can be adequately selected from among oral administration or parenteral administration such as subcutaneous, intravenous, local, transdermal, and enteral (intrarectal) administration, depending on the type of a test substance to be administered.

The present disclosure further provides a method for evaluating hepatotoxicity of a test substance against human hepatocytes, with the use of the chimeric mouse of the present disclosure. An example of this method is an evaluation method comprising the following steps of: (a) administering a test substance to the chimeric mouse of the present disclosure; (b) measuring one or more values in the chimeric mouse to which the test substance is administered in (a); and (c) evaluating the effect of the test substance on human hepatocytes using one or more indicators measured in (b), compared with the one or more indicators of the chimeric mouse to which no test substance is administered.

Preferably, the one or more values are selected from the group consisting of the human albumin concentration, the body weight curve, the liver-weight-to-body-weight ratio, the total albumin level, the total protein level, the ALT level, the AST level, and the total bilirubin level. Preferably, the one or more indicators are selected from the group consisting of an increase or a decrease in any one or more of the human albumin concentration, the body weight curve, the liver-weight-to-body-weight ratio, the total albumin level, the total protein level, the ALT level, the AST level, and the total bilirubin level.

A human nucleic sequence encoding an exemplary Por gene of the disclosure consist or comprises, Genbank Accession number: NM_000941.2:

(SEQ ID NO: 24) 1 gaaggcggtg gtagcgcctc agtggtgtgg gcctgagccc tgcccaggtg cccgcagaga 61 gcagccgggc tgccagcgtt tcatgatcaa catgggagac tcccacgtgg acaccagctc  121 caccgtgtcc gaggcggtgg ccgaagaagt atctcttttc agcatgacgg acatgattct 181 gttttcgctc atcgtgggtc tcctaaccta ctggttcctc ttcagaaaga aaaaagaaga 241 agtccccgag ttcaccaaaa ttcagacatt gacctcctct gtcagagaga gcagctttgt 301 ggaaaagatg aagaaaacgg ggaggaacat catcgtgttc tacggctccc agacggggac 361 tgcagaggag tttgccaacc gcctgtccaa ggacgcccac cgctacggga tgcgaggcat 421 gtcagcggac cctgaggagt atgacctggc cgacctgagc agcctgccag agatcgacaa 481 cgccctggtg gttttctgca tggccaccta cggtgaggga gaccccaccg acaatgccca 541 ggacttctac gactggctgc aggagacaga cgtggatctc tctggggtca agttcgcggt 601 gtttggtctt gggaacaaga cctacgagca cttcaatgcc atgggcaagt acgtggacaa 661 gcggctggag cagctcggcg cccagcgcat ctttgagctg gggttgggcg acgacgatgg 721 gaacttggag gaggacttca tcacctggcg agagcagttc tggccggccg tgtgtgaaca 781 ctttggggtg gaagccactg gcgaggagtc cagcattcgc cagtacgagc ttgtggtcca 841 caccgacata gatgcggcca aggtgtacat gggggagatg ggccggctga agagctacga 901 gaaccagaag cccccctttg atgccaagaa tccgttcctg gctgcagtca ccaccaaccg 961 gaagctgaac cagggaaccg agcgccacct catgcacctg gaattggaca tctcggactc 1021 caaaatcagg tatgaatctg gggaccacgt ggctgtgtac ccagccaacg actctgctct 1081 cgtcaaccag ctgggcaaaa tcctgggtgc cgacctggac gtcgtcatgt ccctgaacaa 1141 cctggatgag gagtccaaca agaagcaccc attcccgtgc cctacgtcct accgcacggc 1201 cctcacctac tacctggaca tcaccaaccc gccgcgtacc aacgtgctgt acgagctggc 1261 gcagtacgcc tcggagccct cggagcagga gctgctgcgc aagatggcct cctcctccgg 1321 cgagggcaag gagctgtacc tgagctgggt ggtggaggcc cggaggcaca tcctggccat 1381 cctgcaggac tgcccgtccc tgcggccccc catcgaccac ctgtgtgagc tgctgccgcg 1441 cctgcaggcc cgctactact ccatcgcctc atcctccaag gtccacccca actctgtgca 1501 catctgtgcg gtggttgtgg agtacgagac caaggctggc cgcatcaaca agggcgtggc 1561 caccaactgg ctgcgggcca aggagcctgc cggggagaac ggcggccgtg cgctggtgcc 1621 catgttcgtg cgcaagtccc agttccgcct gcccttcaag gccaccacgc ctgtcatcat 1681 ggtgggcccc ggcaccgggg tggcaccctt cataggcttc atccaggagc gggcctggct 1741 gcgacagcag ggcaaggagg tgggggagac gctgctgtac tacggctgcc gccgctcgga 1801 tgaggactac ctgtaccggg aggagctggc gcagttccac agggacggtg cgctcaccca 1861 gctcaacgtg gccttctccc gggagcagtc ccacaaggtc tacgtccagc acctgctaaa 1921 gcaagaccga gagcacctgt ggaagttgat cgaaggcggt gcccacatct acgtctgtgg 1981 ggatgcacgg aacatggcca gggatgtgca gaacaccttc tacgacatcg tggctgagct 2041 cggggccatg gagcacgcgc aggcggtgga ctacatcaag aaactgatga ccaagggccg 2101 ctactccctg gacgtgtgga gctaggggcc tgcctgcccc acccacccca cagactccgg 2161 cctgtaatca gctctcctgg ctccctcccg tagtctcctg ggtgtgtttg gcttggcctt 2221 ggcatgggcg caggcccagt gacaaagact cctctgggcc tggggtgcat cctcctcagc 2281 ccccaggcca ggtgaggtcc accggcccct ggcagcacag cccagggcct gcatgggggc 2341 accgggctcc atgcctctgg aggcctctgg ccctcggtgg ctgcacagaa gggctctttc 2401 tctctgctga gctgggccca gcccctccac gtgatttcca gtgagtgtaa ataattttaa 2461 ataacctctg gcccttggaa taaagttctg ttttctgtaa aaaaaaaaa 

The corresponding human amino acid sequence encoding an exemplary Por gene of the disclosure consist or comprises, Genbank Accession number: NP_000932.3:

(SEQ ID NO: 25) 1 minmgdshvd tsstvseava eevslfsmtd milfslivgl ltywflfrkk keevpeftki 61 qtltssvres sfvekmkktg rniivfygsq tgtaeefanr lskdahrygm rgmsadpeey 121 dladlsslpe idnalvvfcm atygegdptd naqdfydwlq etdvdlsgvk favfglgnkt 181 yehfnamgky vdkrleqlga qrifelglgd ddgnleedfi twreqfwpav cehfgveatg 241 eessirqyel vvhtdidaak vymgemgrlk syenqkppfd aknpflaavt tnrklnqgte 301 rhlmhleldi sdskiryesg dhvavypand salvnqlgki lgadldvvms lnnldeesnk 361 khpfpcptsy rtaltyyldi tnpprtnvly elaqyaseps eqellrkmas ssgegkelyl 421 swvvearrhi lailqdcpsl rppidhlcel lprlqaryys iassskvhpn svhicavvve 481 yetkagrink gvatnwlrak epagenggra lvpmfvrksq frlpfkattp vimvgpgtgv 541 apfigfiger awlrqqgkev getllyygcr rsdedylyre elaqfhrdga ltqlnvafsr 601 eqshkvyvqh llkqdrehlw klieggahiy vcgdarnmar dvqntfydiv aelgamehaq 661 avdyikklmt kgrysldvws 

A murine nucleic sequence encoding an exemplary Por gene of the disclosure consist or comprises, Genbank Accession number: NM_008898.2:

(SEQ ID NO: 26) 1 gggccgtggt agcgcctcag tggtgcgggc ttgcgtccgg ccccagtgcc tcagagacct 61 acaggaccgc gcgcggtgtg tgatctggtc ggtaccgagg agcgcaggtt gtgtcaccaa 121 catgggggac tctcacgaag acaccagtgc cacagtgcct gaggcagtgg ctgaagaagt 181 gtctctattc agcacaacgg acattgttct gttttctctc atcgtggggg tcctgaccta 241 ctggttcatc tttaaaaaga agaaagaaga gataccggag ttcagcaaga tccagacaac 301 ggccccacct gtcaaagaga gcagcttcgt ggaaaagatg aagaaaacgg gaaggaacat 361 tattgtattc tatggctccc agacgggaac cgcggaggag tttgccaacc ggctgtccaa 421 ggatgcccac cgctatggga tgcggggcat gtctgcagac cctgaagagt atgacttggc 481 cgacctgagc agcctgcctg agatcgacaa gtccctggta gtcttctgca tggccacata 541 cggagaaggc gaccccaccg acaacgcgca ggacttctat gattggctgc aggagactga 601 cgtggacctc acgggtgtca agtttgctgt gtttggtctc gggaacaaga cctatgagca 661 cttcaacgcc atgggcaagt atgtggacca gcggctggag cagcttggcg cccagcgaat 721 ctttgagttg ggccttggtg atgacgacgg gaacttggaa gaggatttca tcacatggag 781 ggagcagttc tggccagctg tgtgcgagtt cttcggggtg gaagccactg gggaggagtc 841 gagcatccgc cagtacgagc tcgtggtcca cgaagacatg gacacagcca aggtgtacac 901 gggtgagatg ggccgtctga agagctacga gaaccagaaa ccccccttcg atgccaagaa 961 tccattcctg gctgctgtca ccacgaaccg gaagctgaac caaggcactg agaggcatct 1021 aatgcacctg gaattggaca tctcagactc caagatcagg tatgaatctg gagatcacgt 1081 ggctgtgtac ccagccaacg actccaccct ggtcaaccag attggggaga tcctgggggc 1141 tgacctggat gtcatcatgt ctctaaacaa tctcgatgag gagtcgaata agaagcatcc 1201 gttcccctgc cccaccacct accgcacggc cctcacctac tacctggaca tcactaaccc 1261 gccacgaacc aacgtgctct acgagctggc ccagtacgcc tcagagccct cggagcagga 1321 acacctgcac aagatggcgt cctcctccgg cgagggcaag gagctgtacc tgagctgggt 1381 ggtggaggcc cggaggcaca tcctagccat tctccaagac tacccgtccc tgcggccacc 1441 catcgaccac ctgtgcgagc tcctcccgag gctgcaggcc cgctactatt ccattgcctc 1501 gtcgtctaag gtccacccca actccgtgca catctgcgcc gtggctgtgg agtatgaagc 1561 gaagtctgga cgagtgaaca agggggtggc caccagctgg cttcggacca aggaaccagc 1621 aggagagaat ggccgccggg ccctggtccc catgttcgtc cgcaagtccc agttccgctt 1681 gcctttcaag cccaccacac ctgttatcat ggtgggcccc ggcactgggg ttgccccttt 1741 catgggcttc atccaggagc gggcttggct tcgagagcaa ggcaaggagg tcggagagac 1801 gctgctctac tacggctgcc ggcgctcgga tgaggactat ctgtaccgcg aggagctggc 1861 gcgcttccac aaggacggcg ccctcacgca gcttaatgtg gccttttccc gtgagcaggc 1921 ccacaaggtc tatgttcagc acctgctcaa gagggacaaa gagcacctgt ggaagctgat 1981 ccacgaaggt ggtgcccaca tctatgtctg cggggatgct cgaaatatgg ccaaagatgt 2041 gcagaacaca ttctatgaca tcgtggccga gtttgggccc atggagcaca cccaggctgt 2101 ggactatgtt aagaagctca tgaccaaggg ccgctactcg ctggatgtat ggagctagga 2161 gctgccgccc ccacctctgg gctccctgta atcacgtcct taacttcctt ctgccgacct 2221 cccacccctc tggttcctgc cctgcctgga cacagggagg cccagggact gactcctggc 2281 ctgagtgatg ccctcctggg cccttaggca gagcctggtc cattgtacca ggcagcctag 2341 cccagcccag ggcacatggc aagagggact ggacccacct ttgggtgatg ggtgccttag 2401 gtccccagca gctgtacaga aggggctctt ctctccacag agctggggtg cagccccaac 2461 atgtgatttt gaatgagtgt aaataatttt aaataacctg gcccttggaa taaagttgtt 2521 ttctgta

The corresponding murine amino acid sequence encoding an exemplary Por gene of the disclosure consist or comprises, Genbank Accession number: NP_032924.1:

(SEQ ID NO: 27) 1 mgdshedtsa tvpeavaeev slfsttdivl fslivgvlty wfifkkkkee ipefskiqtt 61 appvkessfv ekmkktgrni ivfygsqtgt aeefanrlsk dahrygmrgm sadpeeydla 121 dlsslpeidk slvvfcmaty gegdptdnaq dfydwlqetd vdltgvkfav fglgnktyeh 181 fnamgkyvdq rleqlgagri felglgdddg nleedfitwr eqfwpavcef fgveatgees 241 sirqyelvvh edmdtakvyt gemgrlksye nqkppfdakn pflaavttnr klnqgterhl 301 mhleldisds kiryesgdhv avypandstl vnqigeilga dldvimslnn ldeesnkkhp 361 fpcpttyrta ltyylditnp prtnvlyela qyasepsege hlhkmasssg egkelylswv  421 vearrhilai lqdypslrpp idhlcellpr lqaryysias sskvhpnsvh icavaveyea 481 ksgrvnkgva tswlrtkepa gengrralvp mfvrksqfrl pfkpttpvim vgpgtgvapf 541 mgfiqerawl reqgkevget llyygerrsd edylyreela rfhkdgaltq lnvafsreqa 601 hkvyvqhllk rdkehlwkli heggahiyvc gdarnmakdv qntfydivae fgpmehtqav 661 dyvkklmtkg rysldvws

A human nucleic sequence encoding an exemplary II2-rg gene of the disclosure consist or comprises, Genbank Accession number: NM_000206.2:

(SEQ ID NO: 28)    1 agaggaaacg tgtgggtggg gaggggtagt gggtgaggga cccaggttcc tgacacagac   61 agactacacc cagggaatga agagcaagcg ccatgttgaa gccatcatta ccattcacat  121 ccctcttatt cctgcagctg cccctgctgg gagtggggct gaacacgaca attctgacgc  181 ccaatgggaa tgaagacacc acagctgatt tcttcctgac cactatgccc actgactccc  241 tcagtgtttc cactctgccc ctcccagagg ttcagtgttt tgtgttcaat gtcgagtaca  301 tgaattgcac ttggaacagc agctctgagc cccagcctac caacctcact ctgcattatt  361 ggtacaagaa ctcggataat gataaagtcc agaagtgcag ccactatcta ttctctgaag  421 aaatcacttc tggctgtcag ttgcaaaaaa aggagatcca cctctaccaa acatttgttg  481 ttcagctcca ggacccacgg gaacccagga gacaggccac acagatgcta aaactgcaga  541 atctggtgat cccctgggct ccagagaacc taacacttca caaactgagt gaatcccagc  601 tagaactgaa ctggaacaac agattcttga accactgttt ggagcacttg gtgcagtacc  661 ggactgactg ggaccacagc tggactgaac aatcagtgga ttatagacat aagttctcct  721 tgcctagtgt ggatgggcag aaacgctaca cgtttcgtgt tcggagccgc tttaacccac  781 tctgtggaag tgctcagcat tggagtgaat ggagccaccc aatccactgg gggagcaata  841 cttcaaaaga gaatcctttc ctgtttgcat tggaagccgt ggttatctct gttggctcca  901 tgggattgat tatcagcctt ctctgtgtgt atttctggct ggaacggacg atgccccgaa  961 ttcccaccct gaagaaccta gaggatcttg ttactgaata ccacgggaac ttttcggcct 1021 ggagtggtgt gtctaaggga ctggctgaga gtctgcagcc agactacagt gaacgactct 1081 gcctcgtcag tgagattccc ccaaaaggag gggcccttgg ggaggggcct ggggcctccc 1141 catgcaacca gcatagcccc tactgggccc ccccatgtta caccctaaag cctgaaacct 1201 gaaccccaat cctctgacag aagaacccca gggtcctgta gccctaagtg gtactaactt 1261 tccttcattc aacccacctg cgtctcatac tcacctcacc ccactgtggc tgatttggaa 1321 ttttgtgccc ccatgtaagc accccttcat ttggcattcc ccacttgaga attacccttt 1381 tgccccgaac atgtttttct tctccctcag tctggccctt ccttttcgca ggattcttcc 1441 tccctccctc tttccctccc ttcctctttc catctaccct ccgattgttc ctgaaccgat 1501 gagaaataaa gtttctgttg ataatcatca aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

The corresponding human amino acid sequence encoding an exemplary II2-rg gene of the disclosure consist or comprises, Genbank Accession number: NP_000197.1:

(SEQ ID NO: 29)   1 mlkpslpfts llflqlpllg vglnttiltp     ngnedttadf flttmptdsl svstlplpev  61 qcfvfnveym nctwnsssep qptnltlhyw     yknsdndkvq kcshylfsee itsgcqlqkk 121 eihlyqtfvv qlqdpreprr qatqmlklqn     ivipwapenl tlhklsesql elnwnnrfln 181 hclehlvqyr tdwdhswteq svdyrhkfsl     psvdgqkryt frvrsrfnpl cgsaqhwsew 241 shpihwgsnt skenpflfal eavvisvgsm     gliisllcvy fwlertmpri ptlknledlv 361 teyhgnfsaw sgvskglaes lqpdyserlc     lvseippkgg algegpgasp cnqhspywap 301 pcytlkpet

A murine nucleic sequence encoding an exemplary II2-rg gene of the disclosure consist or comprises, Genbank Accession number: NM_013563.4:

(SEQ ID NO: 30)    1 aggaaatgta tgggtgggga gggcttgtgg gagagtggtt cagggttctg acacagacta   61 cacccagaga aagaagagca agcaccatgt tgaaactatt attgtcacct agatccttct  121 tagtccttca gctgctcctg ctgagggcag ggtggagctc caaggtcctc atgtccagtg  181 cgaatgaaga catcaaagct gatttgatcc tgacttctac agcccctgaa cacctcagtg  241 ctcctactct gccccttcca gaggttcagt gctttgtgtt caacatagag tacatgaatt  301 gcacttggaa tagcagttct gagcctcagg caaccaacct cacgctgcac tataggtaca  361 aggtatctga taataataca ttccaggagt gcagtcacta tttgttctcc aaagagatta  421 cttctggctg tcagatacaa aaagaagata tccagctcta ccagacattt gttgtccagc  481 tccaggaccc ccagaaaccc cagaggcgag ctgtacagaa gctaaaccta cagaatcttg  541 tgatcccacg ggctccagaa aatctaacac tcagcaatct gagtgaatcc cagctagagc  601 tgagatggaa aagcagacat attaaagaac gctgtttaca atacttggtg cagtaccgga  661 gcaacagaga tcgaagctgg acggaactaa tagtgaatca tgaacctaga ttctccctgc  721 ctagtgtgga tgagctgaaa cggtacacat ttcgggttcg gagccgctat aacccaatct  781 gtggaagttc tcaacagtgg agtaaatgga gccagcctgt ccactggggg agtcatactg  841 tagaggagaa tccttccttg tttgcactgg aagctgtgct tatccctgtt ggcaccatgg  901 ggttgattat taccctgatc tttgtgtact gttggttgga acgaatgcct ccaattcccc  961 ccatcaagaa tctagaggat ctggttactg aataccaagg gaacttttcg gcctggagtg 1021 gtgtgtctaa agggctgact gagagtctgc agccagacta cagtgaacgg ttctgccacg 1081 tcagcgagat tccccccaaa ggaggggccc taggagaggg gcctggaggt tctccttgca 1141 gcctgcatag cccttactgg cctcccccat gttattctct gaagccggaa gcctgaacat 1201 caatcctttg atggaacctc aaagtcctat agtcctaagt gacgctaacc tcccctactc 1321 Accttggcaa Tctggatcca Atgctcactg Ccttcccttg gggctaagtt tcgatttcct 1261 gtcccatgta actgcttttc tgttccatat gccctacttg agagtgtccc ttgccctctt 1381 tccctgcaca agccctccca tgcccagcct aacacctttc cactttcttt gaagagagtc 1441 ttaccctgta gcccagggtg gctgggagct cactatgtag gccaggttgg cctccaactc 1501 acaggctatc ctcccacctc tgcctcataa gagttggggt tactggcatg caccaccaca 1561 cccagcatgg tccttctctt ttataggatt ctccctccct ttttctacct atgattcaac 1621 tctttccaaa tcaacaagaa ataaagtttt taaccaatga tca

The corresponding murine amino acid sequence encoding an exemplary II2-rg gene of the disclosure consist of, Genbank Accession number: NP_038591.1:

(SEQ ID NO: 31)   1 mlklllsprs flvlqllllr agwsskvlms     sanedikadl iltstapehl saptlplpev  61 qcfvfnieym nctwnsssep qatnltlhyr     ykvsdnntfq ecshylfske itsgcqiqke 121 diglyqtfvv qlqdpqkpqr ravqklnlqn     lviprapenl tlsnlsesql elrwksrhik 181 erclqylvqy rsnrdrswte livnheprfs     lpsvdelkry tfrvrsrynp icgssqqwsk 241 wsqpvhwgsh tveenpslfa leavlipvgt     mgliitlifv ycwlermppi ppiknledlv 301 teyqgnfsaw sgvskgltes lqpdyserfc     hvseippkgg algegpggsp cslhspywpp 361 pcyslkpea

A human nucleic sequence encoding an exemplary Rag2 gene of the disclosure consist or comprises, Genbank Accession number: NM_000536.3:

(SEQ ID NO: 32) 1 attagatcag tcttcataag gccacagtca ggcacacata cacactctct ttacagtcag 61 ccttctgctt aacatctgta tagtgggcag tcagtgaatc ttccccaagt gctgacaatt 121 aatacctggt ttagcggcaa agattcagag aggcgtgagc agcccctctg gccttcagac 181 aaaaatctac gtaccatcag aaactatgtc tctgcagaty gtaacagtca gtaataacat 241 agccttaatt cagccaggct tctcactgat gaattttgat ggacaagttt tcttctttgg 301 acaaaaaggc tcgcccaaaa gatcctgccc cactggagtt ttccatctgg atgtaaagca 361 taaccatgtc aaactgaagc ctacaatttt ctctaaggat tcctgctacc tccctcctct 421 tcgctaccca gccacttgca cattcaaagg cagcttggag tctgaaaagc atcaatacat 481 catccatgga gggaaaacac caaacaatga ggtttcagat aagatttatg tcatgtctat 541 tgtttgcaag aacaacaaaa aggttacttt tcgctgcaca gagaaagact tcgtaggaga 601 tgttcctgaa gccagatatg gtcattccat taatgtggtg tacagccgag ggaaaagtat 661 gggtgttctc tttggaggac gctcatacat gccttctacc cacagaacca cagaaaaatg 721 gaatagtgta gctgactgcc tgccctgtgt tttcctggtg gattttgaat ttgggtgtgc 781 tacatcatac attcttccag aacttcagga tgggctatct tttcatgtct ctattgccaa 841 aaatgacacc atctatattt taggaggaca ttcacttgcc aataatatcc ggcctgccaa 901 cctgtacaga ataagggttg atcttcccct gggtagccca gctgtgaatt gcacagtctt 961 gccaggagga atctctgtct ccagtgcaat cctgactcaa actaacaatg atgaatttgt 1021 tattgttggt ggctatcagc ttgaaaatca aaaaagaatg atctgcaaca tcatctcttt 1081 agaggacaac aagatagaaa ttcgtgagat ggagacccca gattggaccc cagacattaa 1141 gcacagcaag atatggtttg gaagcaacat gggaaatgga actgtttttc ttggcatacc 1201 aggagacaat aaacaagttg tttcagaagg attctatttc tatatgttga aatgtgctga 1261 agatgatact aatgaagagc agacaacatt cacaaacagt caaacatcaa cagaagatcc 1321 aggggattcc actccctttg aagactctga agaattttgt ttcagtgcag aagcaaatag 1381 ttttgatggt gatgatgaat ttgacaccta taatgaagat gatgaagaag atgagtctga 1441 gacaggctac tggattacat gctgccctac ttgtgatgtg gatatcaaca cttgggtacc 1501 attctattca actgagctca acaaacccgc catgatctac tgctctcatg gggatgggca 1561 ctgggtccat gctcagtgca tggatctggc agaacgcaca ctcatccatc tgtcagcagg 1621 aagcaacaag tattactgca atgagcatgt ggagatagca agagctctac acactcccca 1681 aagagtccta cccttaaaaa agcctccaat gaaatccctc cgtaaaaaag gttctggaaa 1741 aatcttgact cctgccaaga aatcctttct tagaaggttg tttgattagt tttgcaaaag 1801 cctttcagat tcaggtgtat ggaatttttg aatctatttt taaaatcata acattgattt 1861 taaaaataca tttttgttta tttaaaatgc ctatgttttc ttttagttac atgaattaag 1921 ggccagaaaa aagtgtttat aatgcaatga taaataaagt cattctagac cctatacatt 1981 ttgaaaatat tttacccaaa tactcaattt actaatttat tcttcactga ggatttctga 2041 tctgattttt tattcaacaa accttaaaca cccagaagca gtaataatca tcgaggtatg 2101 tttatattta ttatataagt cttggtaaca aataacctat aaagtgttta tgacaaattt 2161 agccaataaa gaaattaaca cccaaaagaa ttaaattgat tattttgtgc aacataacaa 2221 ttcggcagtt ggccaaaact taaaagcaag atctactaca tcccacatta gtgttcttta 2281 tataccttca agcaaccctt tcgattatgc ccatgaacaa gttagtttct catagcttta 2341 cagatgtaga tataaatata aatatatgta tacatataga tagataatgt tctccactga 2401 cacaaaagaa gaaataaata atctacatca aaaaaaaaaa aaaaaaaaaa aaaaaaa

The corresponding human amino acid sequence encoding an exemplary Rag2 gene of the disclosure consist or comprises, Genbank Accession number: NP_000527.2:

(SEQ ID NO: 33)   1 mslqmvtvsn nialiqpgfs lmnfdgqvff     fgqkgwpkrs cptgvfhldv khnhvklkpt  61 ifskdscylp plrypatctf kgslesekhq     yiihggktpn nevsdkiyvm sivcknnkkv 121 tfrctekdlv gdvpearygh sinvvysrgk     smgvlfggrs ympsthrtte kwnsvadclp 181 cvflvdfefg catsyilpel qdglsfhvsi     akndtiyilg ghslannirp anlyrirvdl 241 plgspavnct vlpggisvss ailtqtnnde     fvivggyqle nqkrmicnii slednkieir 301 emetpdwtpd ikhskiwfgs nmgngtvflg     ipgdnkqvvs egfyfymlkc aeddtneeqt 361 tftnsqtste dpgdstpfed seefcfsaea     nsfdgddefd tyneddeede setgywitcc 421 ptcdvdintw vpfystelnk pamiycshgd     ghwvhaqcmd laertlihls agsnkyycne 481 hveiaralht pqrvlplkkp pmkslrkkgs     gkiltpakks flrrlfd

A murine nucleic sequence encoding an exemplary Rag2 gene of the disclosure consist or comprises, Genbank Accession number: NM_009020.3:

(SEQ ID NO: 34)    1 actctaccct gcagccttca gcttggcaca aactaaacag tgactcttcc ccaagtgccg   61 agtttaattc ctggcttggc cgaaaggatt cagagaggga taagcagccc ctctggcctt  121 cagtgccaaa ataagaaaga gtatttcaca tccacaagca ggaagtacac ttcatacctc  181 tctaagataa aagacctatt cacaatcaaa aatgtccctg cagatggtaa cagtgggtca  241 taacatagcc ttaattcaac caggcttctc acttatgaat tttgatggcc aagttttctt  301 ctttggccag aaaggctggc ctaagagatc ctgtcctact ggagtctttc attttgatat  361 aaaacaaaat catctcaaac tgaagcctgc aatcttctct aaagattcct gctacctccc  421 acctcttcgt tatccagcta cttgctcata caaaggcagc atagactctg acaagcatca  481 atatatcatt cacggaggga aaacaccaaa caatgagctt tccgataaga tttatatcat  541 gtctgtcgct tccaagaata acaaaaaagt tactttccgt tgcacagaga aagacttagt  601 aggagatgtc cctgaaccca gatacggcca ttccattgac gtggtgtata gtcgagggaa  661 aagcatgggt gttctctttg gaggacgttc atacatgcct tctacccaga gaaccacaga  721 aaaatggaat agtgtagctg actgcctacc ccatgttttc ttgatagatt ttgaatttgg  781 gtgtgctaca tcatatattc tcccagaact tcaggatggg ctgtcttttc atgtttctat  841 tgccagaaac gataccgttt atattttggg aggacactca cttgccagta atatacgccc  901 tgctaacttg tatagaataa gagtggacct tcccctgggt accccagcag tgaattgcac  961 agtcttgcca ggaggaatct ctgtctccag tgcaatcctc actcaaacaa acaatgatga 1021 atttgttatt gtgggtggtt atcagctgga aaatcagaaa aggatggtct gcagccttgt 1081 ctctctaggg gacaacacga ttgaaatcag tgagatggag actcctgact ggacctcaga 1141 tattaagcat agcaaaatat ggtttggaag caacatggga aacgggacta ttttccttgg 1201 cataccagga gacaataagc aggctatgtc agaagcattc tatttctata ctttgagatg 1261 ctctgaagag gatttgagtg aagatcagaa aattgtctcc aacagtcaga catcaacaga 1321 agatcctggg gactccactc cctttgaaga ctcagaggaa ttttgtttca gtgctgaagc 1381 aaccagtttt gatggtgacg atgaatttga cacctacaat gaagatgatg aagatgacga 1441 gtctgtaacc ggctactgga taacatgttg ccctacttgt gatgttgaca tcaatacctg 1501 ggttccgttc tattcaacgg agctcaataa acccgccatg atctattgtt ctcatgggga 1561 tgggcactgg gtacatgccc agtgcatgga tttggaagaa cgcacactca tccacttgtc 1621 agaaggaagc aacaagtatt attgcaatga acatgtacag atagcaagag cattgcaaac 1681 tcccaaaaga aaccccccct tacaaaaacc tccaatgaaa tccctccaca aaaaaggctc 1741 tcggaaagtc ttgactcctg ccaagaaatc cttccttaga agactgtttg attaatttag 1801 caaaagcccc tcagactcag gtatattgct ctctgaatct actttcaatc ataaacatta 1861 ttttgatttt tgtttactga aatctctatg ttatgtttta gttatgtgaa ttaagtgctg 1921 ttgtgattta ttgttaagta taactattct aatgtgtgtt ttttaacatc ttatccagga 1981 atgtcttaaa tgagaaatgt tatacagttt tccattaagg atatcagtga taaagtatag 2041 aactcttaca ttattttgta acaatctaca tattgaatag taactaaata ccaataaata 2101 aactaatgca caaaaagtta agttcttttg tgtaataagt agcctatagt tcgtttaaac 2161 agttaaaacc aacagctata tcccacacta ctgctgttta taaattttaa ggtggcctct 2221 ggtttatact tatgagcaga attatatata ttggtcaata ccatgaagaa aaatttaatt 2281 ctatatcaag ccaggcatgg tgatggtgat acatgcctgt aatcctggca cttaggaagt 2341 ggaagaagga agtttgtgag tttgatgctt gttgaggtat gaccttttgc tatgtattgt 2401 agtgtatgag ccccaagacc tgcttgaccc agagacaaga gagtccacac atagatccaa 2461 gtaatgctat gtgaccttgc cccccggtta cttgtgatta ggtgaataaa gatgtcaaca 2521 gccaatagct gggcagaaga gccaaaagtg gggattgagg gtaccctggc ttgatgtagg 2581 aggagaccat gaggaaaggg gagaaaaaag tgatggagga ggagaaagat gccatgagct 2641 aggagttaag aaagcatggc catgagtgct ggccaattgg agttaagagc agcccagatg 2701 aaacatagta agtaataact cagggttatc gatagaaaat agattttagt gccgtactct 2761 ccccagccct agagctgact atggcttact gtaaatataa agtttgtatg tgtcttttat 2821 ccaggaacta aatggtcaaa ggtggagtag aaactctgga ttgggattaa atttttctac 2881 aacaaatgct ggcctgggct agattttatc tcatatccga aggctgacag aacacagagc 2941 actggtaaca ttgccacctg ccatgcacaa agacctgagt ctaatactgt ggacattttc 3001 ttgaagtatc tacatgtact tctggagtga aaacatattc caacaatatg cctttgttta 3061 aatcactcac tcactttggg ccctcacatt atatcctttc aaaatcaatg gttcacccct 3121 ttgaaaatgc ttagccatag tccctcatct tccttaaaga cagttgtcat ctctggaaat 3181 agtcacatgt cattcaaggt ccaatactgt gcagctctga agtatggcat taccacttta 3241 agtgaaaagt gaaatatgaa catgagctca gacaaaggtt tgggactatc actctcaagg 3301 aggctctact gctaagtcct gaactgcttt cacatgaata cagaaattat aacaaaaaat 3361 atgtaatcaa taaaaagaaa actttcatat tcc

The corresponding amino acid sequence encoding an exemplary Rag2 gene of the disclosure consist or comprises of gene consist of, Genbank Accession number: NP_033046.1:

(SEQ ID NO: 35)   1 mslqmvtvgh nialiqpgfs lmnfdgqvff     fgqkgwpkrs cptgvfhfdi kqnhlklkpa  61 ifskdscylp plrypatcsy kgsidsdkhq     yiihggktpn nelsdkiyim svacknnkkv 121 tfrctekdlv gdvpeprygh sidvvysrgk     smgvlfggrs ympstqrtte kwnsvadclp 181 hvflidfefg catsyilpel qdglsfhvsi     arndtvyilg ghslasnirp anlyrirvdl 241 plgtpavnct vlpggisvss ailtqtnnde     fvivggyqle nqkrmvcslv slgdntieis 301 emetpdwtsd ikhskiwfgs nmgngtiflg     ipgdnkqams eafyfytlrc seedlsedqk 361 ivsnsqtste dpgdstpfed seefcfsaea     tsfdgddefd tyneddedde svtgywitcc 421 ptcdvdintw vpfystelnk pamiycshgd     ghwvhaqcmd leertlihls egsnkyycne 481 hvqiaralqt pkrnpplqkp pmkslhkkgs     gkvltpakks flrrlfd

A human nucleic sequence encoding an exemplary Fah gene of the disclosure consist or comprises of gene consist of, Genbank Accession number: NM_000137.2:

(SEQ ID NO: 36)    1 gagaccaaaa gtcaggtagg agcctccggg      gtccctgctg tgtcacccgg acaggccgtg   61 ggggcgggca ggggggcggg gccgggcctg      accacagcgg ccgagttcag tcctgctctc  121 cgcacgccac cttaggcccg cagccgtgcc      gggtgctctt cagcatgtcc ttcatcccgg  181 tggccgagga ttccgacttc cccatccaca      acctgcccta cggcgtcttc tcgaccagag  241 gcgacccaag accgaggata ggtgtggcca      ttggcgacca gatcctggac ctcagcatca  301 tcaagcacct ctttactggt cctgtcctct      ccaaacacca ggatgtcttc aatcagccta  361 cactcaacag cttcatgggc ctgggtcagg      ctgcctggaa ggaggcgaga gtgttcttgc  421 agaacttgct gtctgtgagc caagccaggc      tcagagatga caccgaactt cggaagtgtg  481 cattcatctc ccaggcttct gccacgatgc      accttccagc caccatagga gactacacag  541 acttctattc ctctcggcag catgctacca      acgtcggaat catgttcagg gacaaggaga  601 atgcgttgat gccaaattgg ctgcacttac      cagtgggcta ccatggccgt gcctcctctg  661 tcgtggtgtc tggcacccca atccgaaggc      ccatgggaca gatgaaacct gatgactcta  721 agcctcccgt atatggtgcc tgcaagctct      tggacatgga gctggaaatg gctttttttg  781 taggccctgg aaacagattg ggagagccga      tccccatttc caaggcccat gagcacattt  841 ttggaatggt ccttatgaac gactggagtg      cacgagacat tcagaagtgg gagtatgtcc  901 ctctcgggcc attccttggg aagagttttg      ggaccactgt ctctccgtgg gtggtgccca  961 tggatgctct catgcccttt gctgtgccca      acccgaagca ggaccccagg cccctgccgt 1021 atctgtgcca tgacgagccc tacacatttg      acatcaacct ctctgttaac ctgaaaggag 1081 aaggaatgag ccaggcggct accatatgca      agtccaattt taagtacatg tactggacga 1141 tgctgcagca gctcactcac cactctgtca      acggctgcaa cctgcggccg ggggacctcc 1201 tggcttctgg gaccatcagc gggccggagc      cagaaaactt cggctccatg ttggaactgt 1261 cgtggaaggg aacgaagccc atagacctgg      ggaatggtca gaccaggaag tttctgctgg 1321 acggggatga agtcatcata acagggtact      gccaggggga tggttaccgc atcggctttg 1381 gccagtgtgc tggaaaagtg ctgcctgctc      tcctgccatc atgagatttt ctctgctctt 1441 ctggaaacaa agggctcaag cacccctttc      aaccctgtga ctggggtcct ccctcgggct 1501 gtaggcctgg tccgccattc agtgacaaat      aaagccattg tgctctgagg cctgcactgc 1561 cgcagatgca gctgtgtcca cttatgatcg      tgatttgatc cagtgggtca aggtgtgtaa 1621 agcctccctg ccagatattc attaatatgt      tttctcactc ttattagtga ggtcaggggt 1681 ctttgtggga ttttcttatt agacatccca      ggcctcctgg tattccatgg aatttgaaaa 1801 gagactggca cctgtagtag tcagggctct      ccagagaaat agaaccaagg agaaagaaaa 1741 aaaaaaaaaa

The corresponding human amino acid sequence encoding an exemplary Fah gene of the disclosure consist or comprises of gene consist of, Genbank Accession number: NP_000128.1:

(SEQ ID NO: 37)    1 msfipvaeds dfpihnlpyg vfstrgdprp      rigvaigdqi ldlsiikhlf tgpvlskhqd   61 vfnqptlnsf mglgqaawke arvflqnlls      vsqarlrddt elrkcafisq asatmhlpat  121 igdytdfyss rqhatnvgim frdkenalmp      nwlhlpvgyh grassvvvsg tpirrpmgqm  181 kpddskppvy gacklldmel emaffvgpgn      rlgepipisk ahehifgmvl mndwsardiq  241 kweyvplgpf lgksfgttvs pwvvpmdalm      pfavpnpkqd prplpylchd epytfdinls  301 vnlkgegmsq aaticksnfk ymywtmlqql      thhsvngcnl rpgdllasgt isgpepenfg  361 smlelswkgt kpidlgngqt rkflldgdev      iitgycqgdg yrigfgqcag kvlpallps (SEQ ID NO: 38)    1 gggtgctaaa agaatcacta gggtggggag      gcggtcccag tggggcgggt aggggtgtgt   61 gccaggtggt accgggtatt ggctggagga      agggcagccc ggggttcggg gcggtccctg  121 aatctaaagg ccctcggcta gtctgatcct      tgccctaagc atagtcccgt tagccaaccc  181 cctacccgcc gtgggctctg ctgcccggtg      ctcgtcagca tgtcctttat tccagtggcc  241 gaggactccg actttcccat ccaaaacctg      ccctatggtg ttttctccac tcaaagcaac  301 ccaaagccac ggattggtgt agccatcggt      gaccagatct tggacctgag tgtcattaaa  361 cacctcttta ccggacctgc cctttccaaa      catcaacatg tcttcgatga gacaactctc  421 aataacttca tgggtctggg tcaagctgca      tggaaggagg caagagcatc cttacagaac  481 ttactgtctg ccagccaagc ccggctcaga      gatgacaagg agcttcggca gcgtgcattc  541 acctcccagg cttctgcgac aatgcacctt      cctgctacca taggagacta cacggacttc  601 tactcttctc ggcagcatgc caccaatgtt      ggcattatgt tcagaggcaa ggagaatgcg  661 ctgttgccaa attggctcca cttacctgtg      ggataccatg gccgagcttc ctccattgtg  721 gtatctggaa ccccgattcg aagacccatg      gggcagatga gacctgataa ctcaaagcct  781 cctgtgtatg gtgcctgcag actcttagac      atggagttgg aaatggcttt cttcgtaggc  841 cctgggaaca gattcggaga gccaatcccc      atttccaaag cccatgaaca cattttcggg  901 atggtcctca tgaacgactg gagcgcacga      gacatccagc aatgggagta cgtcccactt  961 gggccattcc tggggaaaag ctttggaacc      acaatctccc cgtgggtggt gcctatggat 1021 gccctcatgc cctttgtggt gccaaaccca      aagcaggacc ccaagccctt gccatatctc 1081 tgccacagcc agccctacac atttgatatc      aacctgtctg tctctttgaa aggagaagga 1141 atgagccagg cggctaccat ctgcaggtct      aactttaagc acatgtactg gaccatgctg 1201 cagcaactca cacaccactc tcttaatgga      tgcaacctga gacctgggga cctcttggct 1261 tctggaacca tcagtggatc agaccctgaa      agctttggct ccatgctgga actgtcctgg 1321 aagggaacaa aggccatcga tgtggagcag      gggcagacca ggaccttcct gctggacggc 1381 gatgaagtca tcataacagg tcactgccag      ggggacggct accgtgttgg ctttggccag 1441 tgtgctggga aagtgctgcc tgccctttca      ccagcctgaa gctccggaag tcacaagaca 1501 cacccttgcc ttatgaggat catgctacca      ctgcatcagt caggaatgaa taaagctact 1561 ttgattgtgg gaaatgccac agaaaaaaaa      aaaaaaa

The corresponding murine amino acid sequence encoding an exemplary Fah gene of the disclosure consist or comprises of gene consist of, Genbank Accession number: NP_034306.2:

(SEQ ID NO: 39)   1 msfipvaeds dfpiqnlpyg vfstqsnpkp     rigvaigdqi ldlsvikhlf tgpalskhqh  61 vfdettlnnf mglgqaawke araslqnlls     asqarlrddk elrgraftsq asatmhlpat 121 igdytdfyss rqhatnvgim frgkenallp     nwlhlpvgyh grassivvsg tpirrpmgqm 181 rpdnskppvy gacrlldmel emaffvgpgn     rfgepipisk ahehifgmvl mndwsardiq 241 qweyvplgpf lgksfgttis pwvvpmdalm     pfvvpnpkqd pkplpylchs qpytfdinls 301 vslkgegmsq aaticrsnfk hmywtmlqql     thhsvngcnl rpgdllasgt isgsdpesfg 361 smlelswkgt kaidveqgqt rtflldgdev     iitghcqgdg yrvgfgqcag kvlpalspa

The following examples are provided to better illustrate the claimed disclosure and are not to be interpreted as limiting the scope of the disclosure. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the disclosure. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the disclosure.

EXAMPLES Example 1: Generation of the Por-Floxed Mouse Strain

Por knock-out first targeting vector was purchased from the National Institutes of Health (NIH) Knock-Out Mouse Program (KOMP) (FIG. 4A). The vector was linearization with the AsisI restriction enzyme, and DNA was electroporated into Jm8A3 mouse embryonic stem cells (ESC) (Pettitt, S. J. et al. “Agouti C57BL/6N embryonic stem cells for mouse genetic resources.” Nat Methods 6, 493-495 (2009)) by the Mouse Embryonic Stem Cell Core at Baylor College of Medicine. Integrated clones were selected using neomycin resistance. DNA of ESC clones was digested with NSiI restriction enzyme and screened for site specific integration by Southern blotting using DIG nonisotopic detection system (Roche Applied Biosciences) following the manufacturer's instructions (full blots in FIG. 17 ). The 500 bp-size 5′ and 3′ probes that bind outside the vector's homology arms were synthesized using the following set of primers.

-   -   5′ POR Fw2 GGCCTCAGAGAGGACATAGTGCCC (SEQ ID NO:1)     -   5′ POR Rev2 GCCCTCTGGTGTCAGGTCCC (SEQ ID NO:2)     -   3′ POR Fw2 CCTCACGCAGCTTAATGTGGCC (SEQ ID NO:3)     -   3′POR Rev2 GGAAGTTAAGGACGTGATTACAGGGAGC (SEQ ID NO:4)

Correctly targeted ESCs cells were injected into C57/BL blastocysts by the Genetically Engineered Mouse Core at Baylor College of Medicine. The male chimeras were bred with C57/BL albino females (Taconic) to access germline transmission of targeted ESC. To remove the FRT-flanked LacZ and the neomycin cassette and generate a conditional POR knock-out strain, the mice were crossed with a Rosa26-FLPe strain (Farley, F. W., Soriano, P., Steffen, L. S. & Dymecki, S. M. “Widespread recombinase expression using FLPeR (flipper) mice.” Genesis 28, 106-110 (2000)). Genotyping was performed by Transnetyx (Cordova, TN).

Example 2: X-Gal Staining

Embryos and fresh liver sections were fixed in 4% PFA for 1 hour at 4° C. and washed 2×30 min in X-Gal rinse buffer (PBS 1× with 0.02% Igepal and 0.01% deoxycholate) followed by overnight incubation with X-Gal staining solution (PBS 1× with 5 mM K₃Fe(CN)₆, 5 mM K₄Fe(CN)₆, 0.02% Igepal, 0.01% deoxycholate, 2 mM MgCl₂, 5 mM EGTA and 1 mg/ml of fresh X-Gal). Samples were post-fixed overnight in 4% PFA at 4° C.

Example 3: Generating of the PIRF (Por^(c−/c)/Il2rg−/−/Rag2−/−/Fah−/−) Mouse Strain

Six gRNA sequences targeting critical exons of the Rag2, Il2-rg or Fah gene were selected (FIG. 1A, FIG. 6 and FIG. 7 ) using two different online tools (crispr.mit.edu and COSMID) (Cradick, T. J., Qiu, P., Lee, C. M., Fine, E. J. & Bao, G. “COSMID: A Web-based Tool for Identifying and Validating CRISPR/Cas Off-target Sites.” Molecular therapy. Nucleic acids 3, e214 (2014)). Complementary oligonucleotides were annealed and ligated into the DR274 vector (Addgene plasmid #42250) (Hwang, W. Y. et al. “Efficient genome editing in zebrafish using a CRISPR-Cas system.” Nat Biotechnol 31, 227-229 (2013)) using standard molecular cloning techniques with the restriction enzyme BsaI (NEB) and T4 DNA Ligase (NEB). A T7 bacterial promoter sequence was inserted into the pX330-U6-Chimeric_BB-CBh-hSpCas9vector (Addgene plasmid #42230) (Cong, L. et al. “Multiplex genome engineering using CRISPR/Cas systems.” Science 339, 819-823 (2013)) upstream of the Cas9 transcription start site using standard molecular cloning techniques. DR274 vectors were cut using DraI (NEB) and gel purified using the Zymoclean Gel DNA Recovery Kit (Zymo, Cat #11-301). In vitro transcription of sgRNA was performed using the MEGAshortscript T7 Transcription Kit (Life Technologies, AM1354), according to manufacturer's instructions. The resulting RNA was purified using the RNA Clean & Concentrator-5 (Zymo, R1015) and eluted in RNAse-free water. Synthesis was verified by polyacrylamide gel electrophoresis. pX330 (with T7 promoter) was digested with NcoI and NotI, and gel purified. Cas9 mRNA was synthesized from the digested pX330-T7 vector using the mMessage mMachine T7 ULTRA Kit (life tech AM1345), according to the manufacturer's protocol. Poly-adenylation was verified by denaturing agarose gel electrophoresis (1% agarose and 6.6% formaldehyde in MOPS buffer).

Zygotes from Por c/c mice were injected with S. pyogenes Cas9 mRNA (60 ng/ul) and the six gRNA (15 ng/uL each). All viable zygotes were implanted into 3 pseudopregnant females. To detect the deleted regions all twenty-three pups were genotyped after weaning using the following primers:

Fah Fw (SEQ ID NO: 5) CTGGGTTGCATACTGGTGGG Fah Rev (SEQ ID NO: 6) AAACAGGGTCTTTGCTGCTG Fah Int Fw (SEQ ID NO: 7) ACAAAGGTGTGGCAAGGGTT I12 Fw (SEQ ID NO: 8) CCACCGGAAGCTACGACAAA I12 Rev (SEQ ID NO: 9) GGGGGAATTGGAGGCATTCT I12 Int Rev (SEQ ID NO: 10) CTTCTTCCCGTGCTACCCTC Rag2 Fw (SEQ ID NO: 11) CCTCCCACCTCTTCGTTATCC Rag2 Rev (SEQ ID NO: 12) AGTCTGAGGGGCTTTTGCTA Rag2 Int Fw (SEQ ID NO: 13) AGTCTGAGGGGCTTTTGCTA

Further offspring genotyping was performed by Transnetyx (Cordova, TN).

Example 4: Humanization of PIRF Mice

Hepatocytes (3×10⁶/mouse) were transplanted into the murine liver of PIRF mice by splenic injections as originally described for mouse hepatocytes (Ponder, K. P. et al. “Mouse hepatocytes migrate to liver parenchyma and function indefinitely after intrasplenic transplantation.” Proc Natl Ac ad Sci USA 88, 1217-1221 (1991)). In brief, the abdominal cavity was opened by a midabdominal incision, and 3×10⁶ human hepatocytes in a volume of 100 μl PBS were injected into the spleen. Immediately after transplantation, selection pressure towards transplanted human hepatocytes was applied by withdrawing the drug nitisinone (NTBC) from the drinking water in the following steps: 2 days at 25%, then 2 days at 12% and eventually 2 days at 6% of the colony maintenance dose (100%=7.5 mg/l) prior to discontinuing the drug completely (Bissig, K. D. et al. “Human liver chimeric mice provide a model for hepatitis B and C virus infection and treatment.” The Journal of clinical investigation 120, 924-930 (2010)). Mice with clinical symptoms (hunched posture, lethargy, weight loss, etc) were put back on 100% nitisinone for a few days before once again being weaned off the drug as described above. In order to determine the extent of human chimerism, human albumin (ELISA, Bethyl laboratories) in the murine blood, having previously shown that human albumin levels correlate with the level of human chimerism assessed by immunostaining of human hepatocytes was measured (Bissig, K. D. et al. (2010)). Only mice with a human chimerism >70% were further used. Where indicated, some PIRF mice were injected intravenously with 100 μl Adenovirus coding CRE recombinase under the CMV promoter (Ad5 CMV-Cre, 2.3×10¹¹ pfu/ml, provided by the Vector Development Laboratory at Baylor College of Medicine) either 24-hours before hepatocyte transplantation and/or when reaching high human chimerism (>70%). Available hepatocyte donor information is given in Table 2. All animal experiments were approved by the Baylor College of Medicine Institutional Animal Care and Use Committee (IACUC). All animals used for humanization (including controls) were female, due to fewer postsurgical complications.

Example 5: qPCR

Total mRNA was isolated from fresh frozen tissue samples using Purelink RNA mini kit (Invitrogen). 2 μg of total mRNA was reverse transcribed using the qScript cDNA supermix (Quanta Biosciences) and 20 ng of cDNA was used for the qPCR reactions, performed with Perfecta SYBR Green Fast Mix (Quanta Biosciences) and analyzed on ABI Prism 7900HT Sequence Detection System (Applied Biosciences). The following primers were used for Por mRNA amplification of PIRF mouse samples:

mPor Fw2 (SEQ ID NO: 14) GGCCCCACCTGTCAAAGAGAGCAGC mPor Rev1: (SEQ ID NO: 15) CAAACTTGACACCCGTGAGGTCC

For humanized PIRF mouse liver samples, mouse Por and human POR were amplified using the following set of primers:

mPor Fw1: (SEQ ID NO: 16) TCTATGGCTCCCAGACGGGAACC mPor Rev2: (SEQ ID NO: 17) CCAATCATAGAAGTCCTGCGCG hPOR Fw1: (SEQ ID NO: 18) CCAATCATAGAAGTCCTGCGCG hPOR Rev5: (SEQ ID NO: 19) ACCTTGGCCGCATCTATGTCGG

Each sample was normalized to Gapdh/GADPH as an internal control gene using the following primers:

mGapdh Fw (SEQ ID NO: 20) AGAACATCATCCCTGCATCCA mGapdh Rev (SEQ ID NO: 21) CAGATCCACGACGGACACATT hGAPDH Fw: (SEQ ID NO: 22) CAGAACATCATCCCTGCCTCTAC hGAPDH Rev: (SEQ ID NO: 23) TTGAAGTCAGAGGAGACCACCTG

Example 6: RNA-Seq Libraries

Whole-transcriptome RNA sequencing (RNA-Seq) was performed using total RNA extracted from fresh-frozen liver tissue sampled from all seven liver lobes. Total RNA was isolated using the Purelink RNA mini kit (Invitrogen). Libraries were generated from total RNA according to the manufacturer's recommendation using the TrueSeq Stranded mRNA LT kit (Illumina). The libraries were sequenced on a NextSeq 500 sequencer. The average read per sample was 17 millions. RNA-Seq TPM expression values were calculated with RSEM⁵² (version 1.2.17) using the read aligner Bowtie2⁵³ applied to the combined human and mouse NCBI Refseq (3/21/16) transcriptomes. RNA sequencing data is available from European Nucleotide Archive, ENA accession PRJEB14714. Low-abundance cytochromes (human <20 TPM and mouse <20 TPM) were only compared if one of the experimental groups reached >20TPM. Gene expression has been normalized to three human housekeeping genes and their murine counterparts (PSMB2, PSMB4, RAB7A and VPS2929; Psmb2, Psmb4, Rab7 and Vps29)⁵⁴. RNA-Seq data is available from European Nucleotide Archive, ENA accession code PRJEB14714

Example 7: Western Blot

Western blotting was performed as described previously (Bissig-Choisat, B. et al. “Development and rescue of human familial hypercholesterolaemia in a xenograft mouse model.” Nature communications 6, 7339 (2015)). Tissue from snap frozen liver was homogenized in RIPA buffer (Sigma, cat #R0278-50 ml) containing proteases inhibitors (Roche, cat #04693159001). 30 μg of total protein was electrophoresed in a NuPAGE 4-12% Bis Tris Gel (Invitrogen, cat #NP0336BOX) and transferred to a PVDF membrane (Millipore, cat #IPVH00010). The blot was then blocked in 5% milk, followed by primary antibody incubation. Rabbit anti-Por (Abcam cat #ab13513) or mouse anti-β-actin (Sigma cat #A1978) were diluted 1:1,000 and 1:3,000, respectively (full blots in FIG. 18 and FIG. 19 ). Secondary antibodies were donkey anti-rabbit IgG/HRP and donkey anti-mouse IgG/HRP (Jackson Immunoresearch Labs, cat #711-035-152 and 711-035-150) used at 1:10,000 and 1:50,000, respectively. The membrane was imaged using Amersham ECL Western Blotting Detection Reagent (General Electric Healthcare Life Sciences, cat #RPN2106).

Example 8: Immunohistochemistry

10 μm sections from cryopreserved tissue blocks were fixed with 3% PFA for 15 minutes and incubated overnight at 4° C. with the following primary antibodies: anti-Por (Abcam, cat #ab13513) diluted 1:500, anti-human Nuclei (EMD Millipore, cat #MAB1281) diluted 1:250 in PBS containing 0.2% Triton X-100 and 0.5% BSA. Secondary antibodies (1:1,000 Alexa-fluor conjugated, Molecular Probes) were incubated for 60 min at room temperature in the same buffer. Sections were mounted with Vectashield plus DAPI (Vector Labs).

Example 9: Mouse Husbandry

All mice (6-10 months old, humanized or non-humanized) were maintained under a standard 12-h dark/light cycle with water and chow provided ad libitum. All animal experiments were approved by the Baylor College of Medicine Institutional Animal Care and Use Committee (IACUC).

Example 10: Sample Preparation for Mass Spectrometry

One group of mice was treated (i.v.) with gefitinib (10 mg/kg) and housed separately in metabolic cages for 16 h feces collection. Feces samples were weighted and homogenized in water (100 mg feces in 1,000 μl of H₂O). Subsequently, 300 μl of methanol was added to 100 μl of the resulting mixture, followed by centrifugation at 15,000 g for 20 min. The supernatant was transferred to a new Eppendorf vial for a second centrifugation (15,000 g for 20 min). The final concentration of agomelatine is 2 μM. Each supernatant was transferred to an auto sampler vial for analysis (described below).

For atazanavir metabolism in liver, liver samples were harvested 30 min after the treatment of atazanavir (i.v., 30 mg/kg). Briefly, livers were weighted and homogenized in water/MeOH with the internal standard agomelatine [100 mg liver in 300 ul of H₂O/MeOH (v/v 3:1)]. Subsequently, 300 μl of methanol was added to 100 μl of the resulting mixture, followed by centrifugation at 15,000 g for 20 min. The supernatant was transferred to a new Eppendorf vial for a second centrifugation (15,000 g for 20 min). The final concentration of agomelatine is 2 μM in samples. Each supernatant was transferred to an auto sampler vial. Five μ1 of each prepared sample was injected to a system combining ultra-high performance liquid chromatography (UHPLC) and quadruple time-of-flight mass spectrometry (QTOFMS) for analysis.

Example 11: Mass Spectrometry (UHPLC-QTOFMS Analyses)

Metabolites from gefitinib and atazanavir were separated using a 1260 Infinity Binary LC System (Agilent Technologies, Santa Clara, CA) equipped with 100 mm×2.7 mm (Agilent XDB C18) column. The column temperature was maintained at 40° C. The flow rate of was 0.3 mL/min with a gradient ranging from 2% to 98% aqueous acetonitrile containing 0.1% formic acid in a 15-min run. Quadrupole time of flight mass spectrometry (QTOFMS) was operated in positive mode with electrospray ionization. Ultra-highly pure nitrogen was applied as the drying gas (12 L/min) and the collision gas. The drying gas temperature was set at 325° C. and nebulizer pressure was kept at 35 psi. The capillary voltages were set at 3.5 kV. During mass spectrometry, real time mass correction and accurate mass were achieved by continuously measuring standard reference ions at m/z 121.0508, 922.0098 in the positive mode. Mass chromatograms and mass spectra were acquired by MassHunter Workstation data Acquisition software (Agilent, Santa Clara, CA) in centroid and profile formats from m/z 50 to 1000. The acquisition rate was set as 1.5 spectra per second. The method used in this study has been validated by the previous study of gefitinib metabolism in human liver microsomes³⁹. Meanwhile, the quality control samples were performed every 10 samples in the process of the sample running. Due to the authentic compounds of metabolites not available, the metabolite identification was based on their exact mass and MS/MS fragments. The chromatograms and relative abundance of metabolite were performed on Qualitative Analysis software (Agilent, Santa Clara, CA). The relative abundance was evaluated based on integrated peak area of each metabolite.

Example 12: Statistics

Sample sizes for experiments were determined by estimated differences between groups and availability of highly humanized mice. No randomization of animals before allocation to experimental groups nor blinding of experimental groups was done. Statistical analysis was performed using PRISM version 6.0 software (Graph Pad software) using Mann-Whitney test, or ANOVA. Statistical significance was assumed with a p-value <0.05 (*). Bars in graphs represent mean±SEM unless noted otherwise. Group size (N) represents biological sample size.

Example 13: Generation of Novel Mouse Model for Hepatocyte Repopulation

In order to functionally block murine cytochrome metabolism, conditional (floxed exon 3 and 4) knock-out of the NADPH-P450 oxidoreductase (Por) gene by targeting mouse embryonic stem cells 28 was generated (FIG. 4 ). Injected blastocysts with properly targeted embryonic stem cells produced chimeras with germline transmission of the Por “knock-out first” allele 29. Expression from the targeted Por locus using the lacZ expression cassette in the embryo and adult liver was confirmed (FIG. 5 ). Next mice were bred with a flippase-expressing strain 30 to generate a CRE recombinase conditional Por knock-out strain (Por^(c/c)). Homozygous zygotes from this strain were injected with the bacterial type II Clustered Regularly-Interspaced Short Palindromic Repeats/Cas9 (CRISPR-Cas9) system^(31, 32, 33) targeting simultaneous deletion of critical exons of the Il2-rg, Rag2, and Fah genes (FIG. 6 and FIG. 7 ) to generate the PIRF strain (FIG. 1A). Homozygous PIRF mice are thus immune-deficient, lacking T, B and NK cells, but are healthy and fertile. Since adenoviral gene therapy vectors efficiently transduce hepatocytes in vivo, the Por gene was deleted using an adenovirus coding the CRE recombinase (Adeno-CRE). Increasing doses (2.2×10⁸⁻¹⁰ per mouse) of the virus were injected intravenously into PIRF mice. Quantitative RT-PCR of the Por mRNA in liver revealed efficient deletion only at high doses of adenovirus (FIG. 1B). Immunostaining for Por (FIG. 1C) confirmed these findings, although a minimal residual signal could be detected by Western blotting even at the highest dose used (FIG. 1D). Por-deleted PIRF mouse livers accumulated lipids starting about two weeks after adenoviral transduction (FIG. 2 ), but in contrast to the immune competent Alb-Cre/Por^(c/c) strain^(25, 27), without infiltration and lacking necrosis (FIG. 20 ). Nevertheless, residual Por expressing hepatocytes had a growth advantage over the lipid-rich Por-deleted hepatocytes, and clonal expansion of a few Por expressing cells could be detected four weeks after adenoviral transduction by immunostaining (FIG. 9 ).

Example 14: Characterization of Humanized PIRF Mice

Human liver chimeric mice using the PIRF strain were generated^(5, 20, 34). To ensure cytochrome P450 metabolism would be human-specific, we injected Adeno-Cre (2.3×10¹⁰ pfu/mouse) before human hepatocyte transplantation and an additional dose of Adeno-Cre in some highly humanized PIRF (Hu-PIRF) mice. Immunostaining revealed that an almost complete deletion of the Por gene could be achieved only in double-injected humanized PIRF (Hu-PIRF 2×) mice (FIG. 2A). Quantitative PCR and Western blotting corroborated the massive reduction of murine Por upon adenoviral delivery of CRE (FIG. 12 ). Gene expression profiling was performed to compare PIRF mice repopulated with human hepatocytes (Hu-PIRF) that were injected with either Adeno-CRE (Hu-PIRF 2×) or Adeno-GFP (FIG. 2B). Both groups were repopulated with human hepatocytes from the same hepatocyte donors (Table 2) to avoid inter-individual variations.

TABLE 2 Characteristics of human hepatocyte donors used in the present disclosure. Hepatocyte Age* Gender Race BMI Cause of death Usage in present study #1 24 Male African American 20.3 Anoxia RNAseq (chimeric mice, hepatocytes) #2 2 Female African American 19.6 Head trauma RNAseq (chimeric mice, hepatocytes) #3 45 Female Caucasian 20.8 Anoxia RNAseq (Chimeric mice) #4 1.2 Female Caucasian 20.8 Head trauma Gefetinib metabolites #5 18 Male Caucasian 24.3 Cardiovascular ATV metabolites *in years

Expression of the murine P450 cytochromes was clearly altered for 27 out 38 genes analyzed after Por deletion (FIG. 2C): 24 cytochromes were significantly upregulated (1.5-12.5-fold) and 3 cytochromes significantly downregulated (0.5-0.3-fold). The expression profiles of these murine cytochromes were by in large comparable to those from previous work in non-humanized, Por-deficient mice (Table 1)³⁵. In the human part of the same chimeric liver, human P450 cytochromes were less altered upon deletion of murine Por (FIG. 2D). Half of the human cytochromes were only slightly altered (0.5-1.5-fold change), while the other half were moderately upregulated (1.5-2.4-fold).

Not all human cytochromes serve an important role in xenobiotic metabolism. From the 200 most-prescribed drugs in the United States, about three-quarter are metabolized through P450 cytochromes, of which CYP3A4/5, 2C9, 2C19, 2D6 and 1A2 contribute to ˜95%³⁶. Comparing these human cytochrome clusters from chimeric livers (Hu-PIRF 2×) with the originating, isogenic primary hepatocytes. For this comparison, two donor hepatocytes (Table 2) and the corresponding human (isogenic) liver chimeric mice (N=6). Expression levels were similar for most clusters, and these important cytochromes were all robustly expressed in chimeric livers (FIG. 2E). Interestingly, some human clusters (CYP1A2, CYP2B6, CYP2C19 and CYP3A4) were expressed at even higher levels in the chimeric liver than in primary human hepatocytes.

Example 15: Xenobiotic Metabolism of Humanized PIRF Mice

To validate Hu-PIRF mice for human drug metabolism, xenobiotic metabolism of gefitinib³⁷, an inhibitor of epidermal growth factor receptor used against lung cancer and a variety of other neoplasia was used³⁸. Gefitinib is metabolized primarily by the P450 cytochrome system, including CYP3A4 and 2D6. Gefitinib metabolites demonstrate considerable differences between human and mouse liver microsomes³⁹, but regardless of dose, route or species, gefitinib is excreted primarily in the feces (less than 7% in the urine)^(40, 41). The feces of non-humanized PIRF mice for gefitinib metabolites during the first 24 hours after intravenous injection of gefitinib was then analyzed.

Mass spectrometry revealed a reduction of several gefitinib metabolites upon deletion of the Por gene, implying a Por-dependent P450 cytochrome deficiency for these metabolites (FIG. 3A and FIG. 13A). Since some metabolites were not significantly altered, the possibility that residual Por activity was responsible for persistent murine P450 cytochrome metabolism system was tested. The Por^(c/c) strain was crossed with a transgenic mouse that expresses CRE under the Albumin promoter. Por protein in the liver of Alb-CRE/Por^(c/c) animals was efficiently deleted (FIG. 14 ); nevertheless, the metabolite profile formed after gefitinib injection was comparable to that of PIRF mice with adenoviral deletion of Por (FIG. 13 ). This result similarity indicates that gefitinib has both P450-dependent and -independent drug metabolism.

The biggest and most relevant reduction was observed for 0-desmethyl gefitinib (M4, M523595), which is by far the most abundant metabolite in human feces. Rodents produce many different metabolites in addition to M4^(40, 41) (FIG. 3B), so the M4 metabolite in murine Por-deleted and Por-expressing humanized and non-humanized control mice was analyzed (FIG. 10 ). The highest levels of M4 were detected in murine Por-deficient Hu-PIRF mice, where human hepatocytes preferentially metabolize gefitinib to M4 and the remaining murine hepatocytes are inhibited in their drug metabolism were used (FIG. 3C). Next, to measure other human-specific metabolites. The most abundant human metabolite was M28, which could not be detected at all in non-humanized control mice. Mass spectrometry again showed the highest level of this human-specific metabolite in murine Por-deficient Hu-PIRF mice (FIG. 3D and FIG. 15 ), confirming that these mice showed liver metabolism more similar to humans.

The Por-deficient Hu-PIRF mouse is a novel model system for drug metabolism studies, and therefore was used to analyze different body compartments, e.g. the serum (one hour after injection) and the urine for these key gefitinib metabolites. M4 could not be detected in the urine and was massively reduced (23-fold in Hu-PIRF mice) in the serum, while M28 was detectable at lower concentrations in both the urine and the serum of Hu-PIRF mice (FIG. 16A and FIG. 16B). Although present at lower levels in both compartments, M28 mirrored relative abundance observed in feces (FIG. 3C). These findings confirm that gefitinib metabolites are primarily excreted trough the feces^(40, 41).

To confirm human xenobiotic metabolism using liver homogenates of PIRF mice. Atazanavir, an antiretroviral drug (protease inhibitor) for treatment of human immunodeficiency virus was tested. Previous studies in human and mouse microsomes demonstrated that atazanavir metabolite M15 is a predominant human metabolite⁴². To determine levels of M15 in humanized PIRF mice, PIRF mice were intravenously injected with atazanavir and their livers harvested, 30 min after injection. M15 levels in Par-deleted humanized PIRF mice were 5.4 times greater than those observed in non-deleted mice (FIG. 3E), again indicating that these mice metabolize drugs as humans do.

Example 16: Deletion of the UDP-Glucose 6-Dehydrogenase (UGDH)

Deletion of the UDP-glucose 6-dehydrogenase (UGDH) leads to depletion of UDP-glucuronate, which is the substrate of all UDP-glucuronosyl transferases (UGT). UGTs glucuronidate lipophilic drugs in the liver (phase II) and thereby contribute to biotransformation of drugs in the liver; glucuronidated drugs are more polar (hydrophilic) and more easily excreted. Deletion of UGDH is embryonically lethal and therefore needs to be deleted conditionally or by somatic genome engineering, similar to POR. Troglitazone was developed as an antidiabetic drug but withdrawn from the market due to hepatotoxicity. Interestingly, mice and humans metabolize the drug differently, meaning that humans mainly generate sulfate metabolites (main circulating metabolites) while glucuronide conjugates of troglitazone are less prevalent in humans. In contrast to mice, which generate mostly glucuronide conjugates. Hence troglitazone offers an opportunity to validate effectiveness of the approach to inhibit UDP-glucuronosyl transferases (UGT) by deletion of UDP-glucose 6-dehydrogenase (UGDH) in human liver chimeric mice in addition to the Por deletion and humanization.

Glutathione synthetase (GSS) catalyzes the second step of glutathione biosynthesis. Glutathione is the substrate of Gluthatione S-transferases (GST), which conjugates the molecule to lipophilic drugs (phase II) and thereby contribute to biotransformation of drugs in the liver.

Somatic genome engineering is used to simultaneously delete murine P450 oxidoreductase (Por) and other murine enzymes involved in drug metabolism in humanized mice. Humanized FRG mice (human albumin in murine serum >2 mg/ml) are injected with Adeno-Associated Virus (AAV, serotype 8) expressing sgRNA targeting an early exon of murine Por, UDP-glucose 6-dehydrogenase (Ugdh) or the glutathione synthetase (Gss) gene (see gene therapy vector design, FIG. 21 ). AAVs are injected (2×10¹¹ GC/AAV/mouse) 1 week before injection of Adenovirus expressing Cas9 (7×10⁹ pfu/Ad/mouse). Control mice are injected with adenoviral vector only (Figure. 22, lower row). Results show a deletion of murine por, as well as ugdh and gss genes. The knockdown by CRISPR/Cas9 of the murine por in humanized mice is substantial but not quite as efficient as the knockdown observed with the loxP/CRE system when looking at the DNA (Figure. 23) and protein levels (Figure. 22). Also, the por deletion particularly in FRG mice is independent of the deletion of the other two genes (ugdh and gss), since their sgRNA (targeting molecule) are all on different AAV vectors. However, immunostaining (FIG. 22 ) demonstrated that substantial amounts of cells had deletion of por and gss (FIG. 22 ), while the deletion of ugdh was less efficient.

Humanized PIRF mouse with transgenic Alb-CRE and deletion of other murine enzymes involved in drug metabolism are used. Por is deleted by expression of CRE, but instead of adenoviral CRE, this PIRF mouse carries an Alb-CRE sequence within the murine genome. These mice efficiently repopulate with human hepatocytes as evidenced by human specific albumin >2 mg/ml in the murine blood and transthyretin (prealbumin) staining in the chimeric liver (Figure. 24). Murine por is efficiently deleted in the liver of these chimeric mice since the albumin promoter is expressed already in late embryonic stages in the liver. Also in these humanized PIRF mice, in addition to the por, gss and ugdh can also be deleted (Figure. 23).

Example 17: Analysis of Troglitazone Metabolites

Troglitazone metabolites (two hours after i.p. injection of 600 mg/kg troglitazone) in the livers of humanized and non-humanized FRG mice with and without Por and Ugdh deletion was analyzed. Non-humanized livers of control mice had much higher amounts of glucuronide conjugates than humans or humanized PIRF mice (Figure. 24). Furthermore, glucuronide conjugates reduced significantly upon deletion of ugdh and por in non-humanized and humanized PIRF mice. This data confirms that deletion of ugdh leads also to a functional impairment or abolishment of UGTs in the human liver chimeric liver.

The present disclosure provides a next generation of humanized mouse model amenable to human drug metabolism with minimal interference from the murine P450 cytochromes. The production of human metabolites for two different drugs between humanized PIRF mice and “normal” humanized FRG mice were compared. Analyses revealed higher concentrations of human metabolites in murine feces and liver homogenate in humanized PIRF mice than in FRG mice and demonstrate that these mice have humanized drug metabolism. The PIRF and FRG strains used in this study are in a mixed (C57B and 129S) genetic background. Aside from potential differences in the background to the two previously published FRG mouse strains^(5, 7), our CRISPR/Cas9 generated knockout strains do not express any transgenes, e.g. the neomycin phosphotransferase that inactivates a wide range of aminoglycoside antibiotics. This model system is useful for early detection of reactive metabolites and is an elegant way to block a large and confounding cluster of drug metabolizing murine enzymes. In addition to the novel mouse model provided herein, the disclosure provides (a) knocking out Por in a combination of multiple organs like the gut and the liver or the lung and the liver would be desirable, (b) additional deletions in other drug-metabolizing enzymes and/or achieving a Por deletion more efficiently. Using transgenic mice expressing Cre recombinase would require yet another crossing step into a quadruple transgenic (PIRF) mouse, however, and an early organ-specific deletion might not generate a robust strain amenable to xenotransplantation.

In summary, the present disclosure provides a novel mouse model combining human chimerism with functional deletion of all murine cytochromes by Por deletion. Such a murine Por-deficient humanization can be used in combination with other repopulation models such as the transgenic uPA mouse^(11, 21). Studies with two different drugs in two different body compartments demonstrate that studies in humanized PIRF mice efficiently identify human metabolites.

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What is claimed is:
 1. A method for preparing a chimeric non-human animal comprising human hepatocytes, the method comprising: (a) providing a non-human animal comprising a reduction or deletion of NADPH-P450 oxidoreductase (Por) gene resulting in reduced or absent expression of Por protein; and (b) transplanting human hepatocytes into the non-human animal.
 2. The method of claim 1, wherein providing the non-human animal comprises reducing or deleting the Por gene resulting in reduced or absent expression of Por protein.
 3. The method of claim 1, wherein the reduced or deleted Por gene is a conditional knockdown or knockout of the Por gene.
 4. The method of claim 1, wherein the reduced or deleted Por gene is the result of a mutation, a transgene, treatment with an exogenous substance or somatic genome engineering.
 5. The method of claim 4, wherein the somatic genome engineering comprises Guide RNA (gRNA) and Caspase
 9. 6. The method of claim 1, wherein the non-human animal comprises a floxed allele of the Por gene, and wherein the non-human animal is provided with a Cre recombinase sufficient to produce a conditional knockout of the Por gene.
 7. The method of claim 6, wherein the non-human animal comprising the floxed allele of the Por gene is provided with at least a first dose of a virus that encodes Cre recombinase.
 8. The method of claim 7, wherein the non-human animal is provided with at least a second dose of a virus that encodes Cre recombinase.
 9. The method of claim 1, comprising (a) providing a non-human animal comprising a floxed allele of the Por gene with a first does of a virus that encodes Cre recombinase; (b) transplanting human hepatocytes into the non-human animal; and (c) providing the non-human animal with a second dose of a virus that encodes Cre recombinase.
 10. The method of claim 9, wherein steps (a) and (b) occur sequentially.
 11. The method of claim 9, wherein steps (a) and (b) occur simultaneously.
 12. The method of claim 7, wherein the non-human animal comprising the floxed allele of the Por gene is crossed with a transgenic non-human animal strain expressing Cre recombinase.
 13. The method of claim 1, wherein the non-human animal further comprises a reduction or deletion of at least one additional gene encoding an enzyme involved in drug metabolism.
 14. The method of claim 13, wherein the at least one additional enzyme is a phase II drug enzyme.
 15. The method of claim 1, wherein the non-human animal further comprises a reduction or deletion of UDP-glucose 6-dehydrogenase (UGDH) gene.
 16. The method of claim 1, wherein the non-human animal further comprises a reduction or deletion of Glutathione synthetase (GSS) gene.
 17. The method of claim 1, wherein the non-human animal is selected from the group consisting of primate, bird, mouse, rat, fowl, dog, cat, cow, horse, goat, camel, sheep and pig.
 18. The method of claim 1, wherein the non-human animal is a mouse.
 19. The method of claim 1, wherein the non-human animal is selected from the group consisting of (i) the FRG (Fah^(−/−)Rag2^(−/−)/Il2rg^(−/−)) non-human animal, (ii) a transgenic urokinase type plasminogen activator (uPA) non-human animal, which overexpress uPA under an inducible promoter, preferably a liver-restricted albumin promoter, (iii) the thymidine kinase-NOD/Shi-scid/IL-2Rγ^(null) (TK-NOG) non-human animal, which is a immunodeficient NOG non-human animal with transgenic expression of thymidine kinase under control of liver-restricted promoter, (iv) a non-human animal expressing an inducible Caspase 8 in the liver, and (v) a non-human animal expressing an inducible Caspase 9 in the liver.
 20. A chimeric non-human animal, offspring thereof, or a portion thereof, which has a chimeric liver comprising human hepatocytes, prepared by the method according to any one of claims 1-19.
 21. The chimeric non-human animal of claim 20, wherein the non-human animal is selected from the group consisting of primate, bird, mouse, rat, fowl, dog, cat, cow, horse, goat, camel, sheep and pig.
 22. The chimeric non-human animal of claim 20, wherein the non-human animal is a mouse.
 23. The chimeric non-human animal of claim 20, wherein chimeric non-human animal substantially lacks autogenous hepatocytes.
 24. The chimeric non-human animal of claim 20, wherein human hepatocytes account for at least 60% of all hepatocytes in the chimeric liver.
 25. The chimeric non-human animal of claim 20, wherein human hepatocytes account for at least 70% of all hepatocytes in the chimeric liver.
 26. The chimeric non-human animal of claim 20, wherein human hepatocytes account for at least 80% of all hepatocytes in the chimeric liver.
 27. The chimeric non-human animal of claim 20, wherein human hepatocytes account for at least 90% of all hepatocytes in the chimeric liver.
 28. The chimeric non-human animal of claim 20, wherein the chimeric non-human animal is immunodeficient.
 29. A method for screening for a substance that affects human liver functions, comprising: (a) administering a test substance to the chimeric non-human animal of claim 20; (b) measuring one or more values in the chimeric non-human animal to which the test substance is administered in (a); and (c) selecting a test substance that causes an increase or decrease in one or more values measured in (b), compared with the one or more values measured in a chimeric non-human animal to which no test substance is administered.
 30. The method of claim 29, wherein the one or more values is selected from the group consisting of a metabolite of the test substance, human albumin concentration, body weight curve, liver-weight-to-body-weight ratio, total albumin level, total protein level, Alanine Aminotransferase (ALT) level, Aspartate Aminotransferase (AST) level, and total bilirubin level, creatinine, Blood Urea Nitrogen (BUN), troponine, blood count, TSH and histological assessment for pathologies in the human and non-human organs.
 31. A method for evaluating the toxicity of a test substance against human hepatocytes and any other non-human organ in the non-human chimeric animal, comprising: (a) administering a test substance to the chimeric non-human animal of claim 20; (b) measuring one or more indicators in the chimeric non-human animal to which the test substance is administered in (a); and (c) evaluating the effect of the test substance on human hepatocytes using, one or more indicators measured in (b), compared with the one or more indicators measured in a chimeric non-human animal to which no test substance is administered.
 32. The method of claim 31, wherein the one or more indicators is selected from the group consisting of an increase or a decrease in any one or more of a metabolite of the test substance, human albumin concentration, body weight curve, liver-weight-to-body-weight ratio, total albumin level, total protein level, ALT level, AST level, and total bilirubin level, creatinine, BUN, troponine, blood count and TSH as well as histological alterations including but not limited to necrosis and apoptosis in the human and non-human organs. 